Long-term stability of root cultures of tomato transformed with Agrobacterium rhizogenes R1601

Tomato explants were transformed with Agrobacter rhizogenesR1601 and root clones used to initiate longterm cultures inliquid and on solid media. The liquid cultures were maintainedfor 50 passages over 25 months in the presence and absence ofkanamycin. During this time the clones retained their high growthrates and antibiotic resistance phenotypes. The nptll gene wasdetected by PCR and dot blot hybridization in DNA from clonesat the 21st passage, and NPT-II enzyme activity was detectedin cell extracts prepared at the 45th passage. The solid cultureswere main tained for 12 passages over 12 months, either withcontinual selection, or with alternation during the last sixpassages between selective and non-selective media. The nptllgene was detected by PCR in DNA from cultures at the 5th passageand NPT-II enzyme activity was detected in cell extracts ofvigorously growing clones from the 12th passage. Passages inthe absence of selection had no discernible effect on the stabilityof the transformed state. The results indicate that the Ri-transformedstate can be maintained in tomato root clones during long periodsof culture. Key words: Agrobacterium rhizogenes, long-term cell culture, root clones, tomato, transformation     

Prorocentrum minimum(clone Exuv) is nutritionally insufficient, but not toxic to the copepod Acartia tonsa

This study tested whether the dinoflagellate Prorocentrum minimum is nutritionally insufficient or toxic to the copepod Acartia tonsa. Experiments were carried out with adult female A. tonsa and the P. minimum clone Exuv, both isolated from Long Island Sound. Initially, the functional and numerical responses of A. tonsa feeding on exponentially growing P. minimum cells were characterized. These experiments revealed that A. tonsa readily ingested P. minimum cells, up to the equivalent of 200% of body carbon day⁻¹, but egg production was relatively low, with a maximum egg production rate of 22% of body carbon day⁻¹. Hence, the egg production efficiency (egg carbon produced versus cell carbon ingested) was low (10%). In a separate experiment, ingestion and egg production rates were measured as a function of food concentration with cells in different growth stages (early-exponential, late-exponential/early-stationary, and late-stationary growth phase) to simulate conditions during a bloom. There was no indication that cells in the stationary phase resulted in lower ingestion or egg production rates relative to actively growing cells. Egg hatching success remained high (>80%) and independent of the cell growth phase. In a third experiment specifically designed to test the hypothesis that P. minimum is toxic, ingestion, egg production and egg hatching success were measured when females were fed mixtures of P. minimum and the diatom Thalassiosira weissflogii, but in which total food concentration was held constant and the proportion of P. minimum in the mixed diet varied. A. tonsa readily ingested P. minimum when it was offered in the mixed diet, with no detrimental effects on egg production or egg hatching observed. Supplementing P. minimum with T. weissflogii increased both the egg production rate and the egg production efficiency. It is concluded that P. minimum is nutritionally insufficient, but not toxic to A. tonsa. Finally, it is estimated that in the field grazing by A. tonsa is approximately equivalent to 30% of the maximum daily growth rate of P. minimum. Hence, copepod grazing cannot be ignored in field and modeling studies of the population dynamics of P. minimum.     

Allelopathic effects of the dinophyte Prorocentrum minimum on the growth of the bacillariophyte Skeletonema costatum

We investigated growth interactions between the dinophyte Prorocentrum minimum and the bacillariophyte Skeletonema costatum using bi-algal cultures under axenic conditions. When low cell densities of P. minimum and high cell densities of S. costatum were inoculated into the same medium, growth of P. minimum was suppressed. Other inoculum combinations resulted in reduced S. costatum maximum cell densities. A mathematical model was used to simulate growth and interactions of P. minimum and S. costatum in bi-algal cultures. The model indicated that P. minimum always outcompeted S. costatum over time. Enriched filtrate from low-density P. minimum cultures significantly stimulated S. costatum growth, but enriched filtrate from high-density P. minimum cultures notably inhibited the growth of S. costatum. Growth of P. minimum was not affected by enriched filtrate from cultures of P. minimum at any density. Filtrates of P. minimum cultures were fractionated by ultrafiltration (molecular weight cutoff >3000 Da), and retentate that included polysaccharide(s) significantly inhibited the growth of S. costatum.     

The Comparative Biology of Closely Related Species Living in the Same Area: V. INTER- AND INTRASPECIFIC INTERFERENCE WITHIN CULTURES OF LEMNA spp. AND SALVINIA NATANS

Studies were made of the growth of populations of Lemna minor,L. polyrrhiza, L. gibba, and Salvinia natans under controlledlaboratory conditions. The intrinsic exponential growth-ratesof the clones were determined in un-crowded cultures, and thechanges in growth-rate of self-crowding cultures were measuredand interpreted in terms of an initial exponential growth-ratefollowed by a phase of arithmetic increase in weight and followedin turn by a phase in which the death of submerged and shadedfronds caused a decline from the arithmetic rate of growth.Mean frond weight declined in self-crowding cultures (exceptof L. gibba). Mixed cultures of two species were examined under self-crowdingconditions and changes in the proportions of the species werefollowed. Whereas the total weight of mixed cultures remainedvery constant between replicates, there was wide variation inthe proportions of components. The variation in the two componentswas most closely correlated (negatively) when the struggle forexistence was most evenly balanced. The mean frond weight ofthe losing component declined during the experiments. The order of decreasing vigour of species measured by variousparameters was as follows: Relative (intrinsic) growth-rate: L. minor > S. natans > L. gibba > L. polyrrhiza Arithmetic growth-rate when crowded: S. natans > L. polyrrhiza > L. gibba > L. minor Asymptotic yield per culture: L. polyrrhiza > L. minor > S. natans > L. gibba Success in mixed cultures: The success of a species in mixture could not be predicted fromthe parameters of growth in pure culture. Morphologic featuressuch as the gibbosity of L. gibba     

Studies on the Growth in Culture of Plant Cells: VIII. THE PRODUCTION OF ETHYLENE BY SUSPENSION CULTURES OF ACER PSEUDOPLATANUS, L.

Sycamore cell suspension cultures in a synthetic medium releaseethylene; during a 24-day incubation period a single culture(initial volume 70 ml) produces c. 4 µ moles. There isa very sharp peak of ethylene production between day 10 andday 14 of culture; at the peak of production c. 2 nmoles ethyleneare released per million cells in 24 h. Evidence is presentedthat 2,4-D enhances ethylene production independently of itseffects on culture growth. Under the standard conditions of culture (250-ml Erlenmeyerflasks closed with aluminium foil and containing 70 ml cellsuspension) the concentration of ethylene in the gas phase ofthe cultures rises above 10 ppm. No evidence was obtained thatthis ethylene is inhibitory to culture growth or that a criticallevel of ethylene is necessary to initiate cell division incultures at a critically low cell density. The low rate of ethylene release by stationary phase culturesis temporarily enhanced by the addition of various solutes andfurther depressed by dilution with water.     

The role of interactions between Prorocentrum minimum and Heterosigma akashiwo in bloom formation

We examined the growth and interactions between the bloom-forming flagellates Prorocentrum minimum and Heterosigma akashiwo using bi-algal culture experiments. When both species were inoculated at high cell densities, growth of H. akashiwo was inhibited by P. minimum. In other combinations of inoculation densities, the species first reaching the stationary phase substantially suppressed maximum cell densities of the other species, but the growth inhibition effect of P. minimum was stronger than that of H. akashiwo. We used a mathematical model to simulate growth and interactions of P. minimum and H. akashiwo in bi-algal cultures. The model indicated that P. minimum always out-competed H. akashiwo over time. Additional experiments showed that crude extracts from P. minimum and H. akashiwo cultures did not affect the growth of either species, but both strongly inhibited the growth of the bloom-forming diatom Skeletonema costatum. Further experiments showed that it was unlikely that reactive oxygen species produced by H. akashiwo were responsible for the inhibition of P. minimum growth.     

Purine Alkaloid Production and Accumulation in Cocoa Callus and Suspension Cultures

Cocoa callus and suspension cultures were found to produce caffeine,theobromine, and theophylline which are typical of the purinealkaloids found in cocoa beans. Production of these purine alkaloidswas monitored in callus cultures for over 2 years and shownto stabilize at concentrations of about 10% those found in vivo.Caffeine and theobromine were produced concomitant with logphase growth of the cultures whilst theophylline productionreached a maximum during stationary phase, reflecting the possiblerole of the latter as a catabolite of caffeine. The effectsof choice of cytokinin, explant tissue, cocoa type, light conditionsand time in culture on purine alkaloid production by callushave been examined. Purine alkaloid production by cocoa suspensioncultures has also been examined and these cultures were shownto be less productive and more variable than callus cultures.The results demonstrate that cocoa tissue cultures can be usefulfor studying secondary metabolism in vitro. Key words: Theobroma cacao, caffeine, theobromine, tissue culture, secondary metabolism     

ECTOCELLULAR GLUCOSIDASE AND PEPTIDASE ACTIVITY OF THE MIXOTROPHIC DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

The activities of the enzymes α‐ and β‐glucosidase, and leucine aminopeptidase were measured in cultures of the dinoflagellate Prorocentrum minimum (Pavill.) J. Schiller and in field samples collected during dinoflagellate blooms occurring in tributaries of the Chesapeake Bay, Maryland, USA. Activities were measured using fluorogenic artificial substrates and partitioned among the >5 μm size fraction, small microbes fraction (0.1–5 μm), and dissolved phase (<0.1 μm). P. minimum and most other photosynthetic dinoflagellates are >5 μm in size and thus can be separated from the small microbes fraction, which contains most bacteria. Little to no glucosidase activity was detected associated with the >5 μm size fraction in cultures or in field samples, with most of the activity (67% to 93% in cultures, 54% to 100% in field samples) in the small microbes size fraction for both α and β glucosidase. In contrast, 67% to 90% of the total leucine aminopeptidase (LAP) activity in cultures was measured in the >5 μm fraction. Within a culture, LAP activity in the size fraction containing P. minimum decreased in response to ammonium and urea additions, but not in response to nitrate. In field samples, LAP activity was positively correlated with dinoflagellate abundance and chl a, and negatively correlated with ammonium concentration. During blooms, up to 34% of LAP activity was associated with the >5 μm fraction, indicating that when abundant, dinoflagellates may make a substantial contribution to ectocellular LAP activity in the water column.     

The correct position of flagellar insertion in Prorocentrum and description of Prorocentrum rhathymum sp. nov. (Pyrrhophyta)

For three Prorocentrum species, P. minimum (Pavillard) Schiller,P. rhathymum sp. nov., and P. triestinum Schiller, we have foundthat both flagella emerge from a single large pore. This arrangementis contrary to previous observations which suggested they arosefrom separate pores. Prorocentrum triestinum has only one anteriorpore, whereas P. minimum and P. rhathymum sp. nov. have an additionalauxiliary pore. The pores in all three species are formed bya series of small plates. The left and right valves on eachspecies can be identified by ornamentation unique to each valve.Prorocentrum rhathymum sp. nov., isolated from the planktonof Cinnamon Bay, Virgin Islands, is described. ² The Biological Laboratories, Harvard University, Cambridge,Massachusetts 02138 ³ Department of Botany, Duke University, Durham, North Carolina27706     

Prorocentrum minimum blooms: potential impacts on dissolved oxygen and Chesapeake Bay oyster settlement and growth

The cosmopolitan dinoflagellate Prorocentrum minimum is a recurrent bloom forming species in the Chesapeake Bay and its tributaries, generally observed at its highest levels in late spring and summer. Laboratory studies were conducted to assess potential bloom impacts on diel oxygen concentrations in shallow littoral zones as well as settlement success and post-set growth of the eastern oyster Crassostrea virginica. Using light–dark and dark cultures and periodic diel sub-sampling, bloom levels of P. minimum produced supersaturated oxygen levels at the end of each day while darkened cultures were typified by rapid decreases in dissolved oxygen (DO) (1.1–1.3 mg L⁻¹ h⁻¹) to hypoxic and anoxic levels within 4 days. These data suggest shallow, poorly flushed systems and the biota in them will experience rapid and large diel variations in oxygen, implying recurrent P. minimum blooms need be considered as short-term oxygen stressors for Bay oyster spat and other living resources. Direct effects of P. minimum impacts on oysters were not as expected or previously reported. In one experiment, pre-bloom isolates of P. minimum were grown and then exposed to polyvinyl chloride (PVC) settlement plates to see whether dinoflagellate preconditioning of the hard substrate might affect oyster sets. No differences were noted between set on the PVC with P. minimum exposure to set recorded with filtered seawater, Instant Ocean^®, or Isochrysis. In the second oyster experiment, spat on PVC plates were exposed to field collected P. minimum blooms and a commercial mixture of several other food types including Isochrysis. Oyster growth was significantly higher in P. minimum exposures than noted in the commercial mix. These results, compared to results with other isolates from the same region, indicate substantial positive impact from some of the P. minimum blooms of the area while others separated in space, time, or nutrient status could severely curtail oyster success through toxin production induced by nutrient limitation.     

The Fate of Sulphate within the Capsular Polysaccharide of the Unicellular Red Alga Rhodella maculata

³⁵SO^2–₄ was used in pulse-chase experiments to study themetabolism of sulphate in the capsular polysaccharides of R.maculata. Radioactivity reached an equilibrium between the chiefintracellular pools during an 8 h pulse and there was no lossof radioactivity from the cells during an extended chase ofup to 14 d, until cultures reached stationary growth phase.Any loss then found was due to dissociation of sulphated capsularmaterial from the cells. The failure of biochemical assays todetect extracellular sulphatase or sulphotransferase activitiessupports the conclusion from pulse-chase experiments that thereis no turnover of sulphate in the capsular polysaccharide ofR. maculata. Key words: sulphated polysaccharides, sulphate turnover, Rhodella     

Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.     

Simultaneous changes in cell quotas of microcystin, chlorophyll a, protein and carbohydrate during different growth phases of a batch culture experiment with Microcystis aeruginosa

The cell quotas of microcystin (Q_mcyst), protein (Q_prot), chlorophylla (Q_chloro) and carbohydrate (Q_carbo), as well as the net productionrates of these parameters, were determined during the exponentialand stationary phases in nine batch cultures of Microcystisaeruginosa (CYA 228) at light regimes from 33 to 53 µmolphotons m^–2 s^–1. The following results were obtained.(i) A parallel pattern was found in the changes of Q_mcyst, Q_prot,Q_chloro and Q_carbo during the entire growth cycle and significantcorrelations were recorded between Q_mcyst and Q_prot, Q_chloroand Q_carbo. (ii) The net microcystin production rate (µ_mcyst)was positively correlated with the specific cell division rate(µ_c), the chlorophyll production rate (µ_chloro)and the protein production rate. (iii) A significant inverselinear relationship was found between µ_c and Q_mcyst, i.e.cultures with a positive µ_c had a Q_mcyst between 110 and400 fg microcystin cell^–1, while declining cultures hadQ_mcyst values >400 fg microcystin cell^–1. Maximum variationin Q_mcyst within cultures was 3.5-fold. Collectively, the resultsshow that cells produced microcystin at rates approximatingthose needed to replace losses to daughter cells during divisionand that microcystin was produced in a similar way to proteinand chlorophyll, indicating a constitutive microcystin production.     

A Possible Role for Urease as a Storage Protein in Aspergillus tamarii

A marked decrease in mycelial urease activity during the endogenousphase of undifferentiated Aspergillus tamariicultures was foundto be independent of preparative procedures but related to thedepletion of external nutrients. The enzyme, which was synthesizedduring the active growth stage, was produced in similar quantitieswith ammonium or urea as sole nitrogen source and at its peakrepresented c. 8·5 per cent of the total soluble proteinpool of the mycelium. It was found to show maximum activityat pH 8·20–8·65 when measured in cell-free,phosphate-buffered extracts. Isolation of urease from differentstages of the endogenous phase by affinity chromatography hasshown that the observed decrease in activity was due to breakdownof the enzyme protein in mature cultures, followed by the progressivedeactivation of residual enzyme during the autolytic stage.Since selective inhibition of 80–90 per cent of activityby acetohydroxamic acid in media containing urea as the onlynitrogen source or total repression of urease synthesis by L-histidinein ammonium-grown cultures did not interfere with normal growth,it was concluded that in A. tamarii urease fulfils the functionof a storage protein with a measure of catalytic activity. Aspergillus tamarii, urease, storage protein, nitrogen metabolism     

Influences of temperature, salinity and irradiance on growth of Prorocentrum minimum (Dinophyceae) from the Mediterranean Sea

A Mediterranean clone of the red-tide forming dinoflagellateProrocentrum minimum was studied in vitro for its capacitiesto adapt to salinity, temperature and light. This clone is euryhalineand shows optimal growth between 15 and 35     

Observations on diurnal vertical migration and phased cell division for three coexisting marine dinoflagellates

An experiment was performed in the beginning of September 1988in the Gullmar Fjord, eastern Skagerrak, in order to study diurnalvertical migration and phased cell division for a natural phytoplanktoncommunity dominated by the dinoflagellates Prorocentrum minimum,Prorocentrum micans and Cerattum furca. A 1 80 m high and 0.30wide non-transparent PVC cylinder was filled with surface watercontaining the dominant dinoflagellates. An artificial haloclinewas created by adding high salinity, nutrient-rich bottom waterto the bottom of the cylinder Cell densities were measured atseven depths at 11 times during 48 h Physical parameters (temperature,salinity, phosphate, nitrate, ammonia and silicate) were measuredat the start and at the end of the experiment in the water columnabove and below the halocline. Strong diurnal vertical migrationpatterns were found for all three species with an aggregationof cells at the surface in the morning and during the day andat the bottom at night. The descent and ascent seemed to startbefore sunset and sunrise respectively and all species wereable to migrate through the strong artificial halocline. Somedifferences were found in the speed and timing of migrationbetween the species with P.minimum having the most pronouncedand fastest migration of the three Cell division frequency washighest at 05.00 at the bottom and also at the surface At othertimes and depths the division was always close to zero. Thedecrease in especially nitrate concentrations in the bottomwater of the cylinder suggests that these dinoflagellates arecapable of dark nitrogen assimilation.     

A laboratory study of the effect of non-phytoplankton diets on the reproduction of Calanus helgolandicus

The effect of five non-phytoplankton and one phytoplankton dietson reproduction is described for the copepod Calanus helgolandicusand compared to field fecundity estimates. A fecundity increasefollowed ingestion of larvae of the sea urchin Sphaerechinusgranularis (LSU) and the oyster Crassostrea gigas (OYS) by copepodfemales, whereas a decrease followed ingestion of eggs of thecopepods Acartia and Temora spp. (COP), as a function of proteinconcentration in diets. With larvae of the sea urchin Paracentrotuslividus (SSU) and the dinoflagellate Prorocentrum minimum (PM;standard phytoplankton diet), fecundity was identical. Althoughprotein concentrations differed greatly from one food treatmentto the other, mean fecundity values were rather low. For unknownreasons, and despite their very high protein concentrationsin comparison to in situ, none of these diets could improvefemale fecundity or reach maximum egg production rates reportedin C.helgolandicus. Hatching rates were >80%, stable andsimilar with both PM and COP diets. With SSU, LSU and OYS diets,egg viability was lower than in situ and with PM, showing astrong and mild decrease with time with LSU and SSU, respectively.Ingestion of zygotes of the brown alga Fucus spiralis (ZYG)led to the death of females. These results suggest that denseblooms of zygotes of macroalgae and invertebrate embryos andlarvae, occurring in the continental shelf as part of copepoddiets, could have different effects on the demographic responseof C.helgolandicus.     

Assimilation numbers in cultures of marine phytoplankton

During exponential growth in batch culture, assimilation numbersof eleven algal species ranged from 1.6–20.8, with a meanvalue of 5.3 g C/g Chlorophyll a/hr. The highest assimilationnumber of 20.8 g C/g Chlorophyll a/hr was observed in Coccolithuspelagicus, due to the relatively low concentration of chlorophylla/cell. The assimilation number declined from exponential tostationary phase in batch cultures for ten algal species, butincreased with age in batch culture in Amphiprora paludasa (abenthic diatom). The assimilation number declined with decreasinggrowth rate in nitrate-limited chemostat cultures of Phaeodactylumtricornutum and in iron-limited chemostat cultures of Phaeodactylumtricornutum and Isochrysis galbana.     

Production of dissolved organic carbon in phyloplankton cultures as measured by high-temperature catalytic oxidation and ultraviolet photo-oxidation methods

The production of dissolved organic carbon (DOC) in culturesof the diatoms Chaetoceros gracilis and Phaeodactylum tricornutum,the flagellate Isochrysis galbana, the dinoflagellate Alexandriumtamarense and a natural algal assemblage from the NorthwestArm, Halifax, Nova Scotia, Canada, was followed using a high-temperaturecatalytic oxidation (HTCO) and a UV photo-oxidation method.Molecular weight fractionation of the DOC was performed fortwo cultures: C.gracilis and I.galbana. While the DOC in theculture medium increased significantly during log-phase growthfor all organisms except the dinoflagellate, this increase wasproportional to the increase in cell numbers; the increase inDOC per cell was either small or zero. In all cultures, maximumrelease took place during stationary and senescent phases, usuallyafter cell numbers had started to decrease. In both C.gracilisand I.galbana, a major portion (>65%) of the organic matterreleased to the medium during log-phase growth had mol. wtsof <10 000 Da. The increase in DOC in the I.galbana culturein stationary and senescent phases was due to the release ofhigh-molecular-weight materials. The differences in extracellularrelease of DOC between species and between different growthstages in the same species suggest that both the species compositionand physiological state of phytoplankton populations must beknown before interpretations and predictions based on fielddata can be made. In order to determine whether the differencesin DOC values found by the HTCO and UV oxidation methods arecaused by the resistance to UV oxidation of some compounds producedby phytoplankton, rather than by less than optimum efficiencyof the UV unit used, standards must be based on naturally occurringcompounds, rather than the pure compounds normally used.     

Dissolved organic carbon released by zooplankton grazing activity-a high-quality substrate pool for bacteria

Experiments were designed to investigate whether processes relatedto zooplankton feeding have a positive effect on bacterial growth.Bacterial abundance and [³H]thymidine incorporation rates werefollowed in grazer-free batch cultures originally containingeither Scenedesmus quadricauda or Rhodomonas lacustris as foodsources, and Daphnia cucullata or Eudiaptomus graciloides asgrazers. Compared with controls lacking either animals or algae,a significantly higher bacterial abundance and productivityoccurred in cultures which contained both phyto- and zoo-plankton.The same experimental methodology was tested during the declineof a diatom spring bloom in a eutrophic, temperate lake. A significantincrease in bacterial biomass was observed due to the grazingactivity of in situ mesozooplankters during the clear-waterphase. Our results demonstrated that the dissolved carbon pathwayfrom mesozooplankton to bacteria averaged 57% (26–78%)of the algal carbon filtered from suspension.