Aphidicolin as synchronizing agent in root tip meristems of Haplopappus gracilis

Aphidicolin, a reversible inhibitor of nuclear DNA replication, was tested as syncrhonizing agent in root tip meristems of Haplopappus gracilis. Embryos (i.e. decoated seeds) or 3-day-old seedlings were used to this purpose. After a 24 h treatment with the drug, a high level of synchrony was observed in both experimental materials for two cell cycles as assessed by determining the accumulation of cells in the S and M phases of the cycle. Highest synchronization was obtained with germinating embryos, possibly owing to a low degree of synchrony already existing in this system.     

Cell cycle arrest induced by theophylline in root meristem of Haplopappus gracilis

Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, induced a block of the cell cycle in G₁, a temporary arrest in G₂ and 70% decrease in the uptake of labelled thymidine in roots of Haplopappus. These effects are compared to those previously found with aminophylline and discussed in view of the possible involvement of cAMP in the regulation of the cell cycle in plants.     

A structural anomaly of the satellite in Haplopappus gracilis

Summary A cytological examination of a population of Haplopappus gracilis revealed one plant possessing a heteromorphic satellited pair of chromosomes in all the root tip cells. The cytology and the detailed morphology of this anomaly are described.     

Occurrence of circadian rhythms in hairy root cultures grown under controlled conditions

Hairy roots obtained by transformation via Agrobacterium rhizogenes provide an artificial plant material devoid of aerial parts with high growth on hormone-free media. Fundamental knowledge of hairy root physiology is essential to develop and control its culture. In contrast to shake-flask cultures, a bioreactor set-up combined with on-line data logging provides an efficient tool to study rapid physiological variations in hairy root cultures. Datura innoxia hairy roots were grown in a bioreactor equipped with on-line data analyses of pH, dissolved oxygen (pO2), conductivity, oxygen, and carbon dioxide. The experiments were done at a constant temperature and in the absence of light cues. The results obtained showed that the carbon dioxide evolution rate (CER) presented regular oscillations during the culture. Similar oscillations were also observed for the oxygen uptake rate (OUR). These signals were treated mathematically to look for the existence of a rhythm. An autocorrelation function was used to detect any periodic components. The results demonstrate that hairy root respiration exhibited peaks of 1 day. These oscillations, having a period of about 24 h, were also observed in pH and conductivity signals, although not for the pO2 signal. The data acquired in the absence of hairy roots showed that the observed periodic behavior was not an artifact. No effect on rhythms was observed by the imposition of an external "day/night" cycle. The fact that oscillations persisted in the absence of external stimuli, with a free-running period of 24 h, suggests that a circadian rhythm exists in hairy roots of D. innoxia.     

Wirkungsspektren der Anthocyansynthese in Gewebekulturen und Keimlingen von Haplopappus gracilis

Summary The biosynthesis of anthocyanin in tissue cultures and intact seedlings of Haplopappus gracilis is a light-dependent reaction which can be induced by blue light only. Anthocyanin appeared in all organs of the seedling.Wounding of the plant led to an increase in the content of anthocyanin due to increased anthocyanin synthesis in the cotyledons.The action spectra of anthocyanin formation in tissue cultures and intact seedlings have two peaks, one at 438 nm and the other at 372 nm. The limit of activity in the direction of longer wavelengths lies between 474 and 493 nm. Red light of short and long wavelength is ineffective in the induction of pigment synthesis. The photoreceptor of the light reaction is supposed to be a yellow pigment (flavoprotein or carotinoid). In contrast to the intact plants, isolated cotyledons and wounded seedlings are able to form anthocyanin not only in the blue region but also during irradiation with red light of high intensity. The action spectrum of anthocyanin synthesis in the isolated cotyledons has a marked maximum at about 440 nm and a second one at about 660 nm. A little activity can be observed throughout the visible spectrum. The pigment synthesis induced by red light can be completely suppressed by DCMU, an inhibitor of photosynthesis. This indicates that in the case of the activity in the red light caused by wounding chlorophyll serves as photoreceptor.The anthocyanin synthesis in tissue cultures and seedlings could not be influenced by low energy radiation in the red or in the far red region, even after induction of anthocyanin synthesis by blue light of high intensity. Therefore it seems that the phytochrome system is not involved in anthocyanin synthesis in Haplopappus gracilis.     

Duration of the Mitotic Cycle in Cell Cultures of Haplopappus gracilis

The duration of the cell cycle and its component phases in cell cultures of Haplopappus gracilis was estimated by means of pulse labelling with tritiated thymidine and subsequent autoradiographic techniques. The total duration of the mitotic cycle was found to be 22.0 hours. The average durations of the following component phases were: the synthetic period (S) 6.4 hours, the postsynthetic period (G₂) 4.86 hours, prophase (P) 0.64 hours, metaphase (M) 0.40 hours, anaphase + early telophase (AT) 0.36 hours, the presynthetic period (G₁) 9.34 hours. The results indicate that G₁ and G₂ are the phases, which are most prolonged in populations of cultivated cells when compared to the same phases in root lip cells from the same species.     

Partial Synchronization of Cell Division in Suspension Cultures of Haplopappus gracilis

Four different chemicals were tested in their ability to synchronize cell division in asynchronous cell cultures of Haplopappus gracilis. Twentyfour-hour treatments with 5-amino uracil resulted in a peak in the mitotic index about 14–16 hours after the end of the treatment. The increase in the frequency of mitoses was about three times that of the control. Hydroxyurea, at a concentration of 3 mM, gave after a treatment period of 12–24 hours an increase in the frequency of mitoses which appeared about 10 hours after the treatment. The mitotic index was about 35 per cent, which is 4 times that of the control. 5-Fluorodeoxyuridine (FUdR) at a concentration of 2 × 10^?7M gave a mitotic burst about 16 hours after treatment. At that time about 15 per cent of the cells were dividing which was about twice that of the control. The block was reversed with 4 × 10^?5M thymidine. Thymidine at a high concentration caused a reduction in the frequency of mitoses during the treatment. After 15 to 16 hours in a thymidine free medium a mitotic peak appeared with a doubling of the frequency of mitoses in treated cells. Cytological studies showed that parlicularly hydroxyurea but also 5-aminouracil and 5-fluorodeoxyuridine produced gaps and fragments at the concentrations which gave cell synchronization.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions

Of bacterial cells in a sample of activated sludge, 34% contained detectable intracellular polyphosphate inclusions following Neisser staining, when grown on glucose/mineral salts medium at pH 5.5; at pH 7.5 only 7% of cells visibly accumulated polyphosphate. In a sludge isolate of Burkholderia cepacia chosen for further study, maximal removal of phosphate and accumulation of polyphosphate occurred at pH 5.5; levels were up to 220% and 330% higher, respectively, than in cells grown at pH 7.5. During the early stationary phase of growth at pH 5.5 a maximum level of intracellular polyphosphate that comprised 13.6% of cellular dry weight was reached. Polyphosphate kinase activity was detected in actively growing cells only when cultured at pH 5.5. The phenomenon of acid-stimulated phosphate uptake and polyphosphate accumulation in this environmental bacterial population parallels observations previously made by us in the yeast Candida humicola and may thus represent a widespread microbial response to low external pH values.     

Modification of reproductive development in Arabidopsis thaliana under spaceflight conditions

Reproductive development in Arabidopsis thaliana (L.) Heynh. cv. Columbia plants was investigated under spaceflight conditions on shuttle mission STS-51. Plants launched just prior to initiation of the reproductive phase developed flowers and siliques during the 10-d flight. Approximately 500 flowers were produced in total by the 12 plants in both the ground control and spaceflight material, and there was no significant difference in the number of flowers in each size class. The flower buds and siliques of the spaceflight plants were not morphologically different from the ground controls. Pollen viability tests immediately post-flight using fluorescein diacetate indicated that about 35% of the pollen was viable in the spaceflight material. Light-microscopy observations on this material showed that the female gametophytes also had developed normally to maturity. However, siliques from the spaceflight plants contained empty, shrunken ovules, and no evidence of pollen transfer to stigmatic papillae was found by light microscopy immediately post-flight or by scanning electron microscopy on fixed material. Short stamen length and indehiscent anthers were observed in the spaceflight material, and a film-like substance inside the anther that connected to the tapetum appeared to restrict the release of pollen from the anthers. These observations indicate that given appropriate growing conditions, early reproductive development in A. thaliana can occur normally under spaceflight conditions. On STS-51, reproductive development aborted due to obstacles in pollination or fertilization.     

An effect of exogenous thymidine on the cell cycle in Haplopappus gracilis

The study of an effect of exogenous thymidine on the mitotic cycle demonstrated that a 30 minute exposure to unlabeled and to tritiated thymidine at a concentration of 2.9 × 10^?6 M was sufficient to cause a significant increase in the mitotic index of root meristem cells of Haplopappus gracilis. An analysis of the data revealed that this was due to the prolongation of metaphase rather than to an increase in the actual number of cells entering division.     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Pollen and ovule development in Arabidopsis thaliana under spaceflight conditions

The development of pollen and ovules in Arabidopsis thaliana on the space shuttle 'Endeavour' (STS-54) was investigated. Plants were grown on nutrient agar for 14 days prior to loading into closed plant growth chambers that received light and temperature control inside the Plant Growth Unit flight hardware on the shuttle middeck. After 6 days in spaceflight the plants were retrieved and immediately dissected and processed for light and electron microscope observation. Reproductive development aborted at an early stage. Pistils were collapsed and ovules inside were seen to he empty. No viable pollen was observed from STS-54 plants; young microspores were deformed and empty. At a late stage, the cytoplasm of the pollen contracted and became disorganized, but the pollen wall developed and the exine appeared normal. The tapetum in the flight flowers degenerated at early stages. Ovules from STS-54 flight plants stopped growing and the integuments and nucellus collapsed and degenerated. The megasporocytes appeared abnormal and rarely underwent meiosis. Apparently they enlarged, or occasionally produced a dyad or tetrad, to assume the form of a female gametophyte with the single nucleus located in an egglike cell that lacks a cell wall. Synergids, polar nuclei, and antipodals were not observed. The results demonstrate the types of lesions occurring in plant reproductive material under spaceflight conditions.     

The biotechnology of hydrogen production by Nostoc flagelliforme grown under chemostat conditions

The potential of using N₂-fixing cyanobacteria to produce hydrogen photobiologically has stimulated research on the physiology and biotechnology of species exhibiting high H₂ production rates over long periods of time. In this work Nostoc flagelliforme, a terrestrial N₂-fixing cyanobacterium, has been examined to establish its physiology and potential for H₂ production under controlled conditions. Cell filaments of N. flagelliforme were purified and grown in liquid culture to optimize its H₂ metabolism. In batch-grown cultures the activity of nitrogenase, the key enzyme for H₂ production in N₂-fixing organisms, was found to be high only during a short phase of exponential growth. A chemostat system was thus constructed for long-term experiments using continuous cultures, with the aim of exploiting the exponential growth phase. The dilution rate (D) and environmental factors, such as N₂ concentration in the gas phase and temperature, significantly influenced H₂ production. Cells grown continuously under the optimized conditions of D = 0.022 h⁻¹, 34 °C and 5.1 kPa N₂ in the gas phase exhibited H₂ production rates that were more than four times higher than the maximal rates under standard batch growth conditions. Received: 14 October 1996 / Received revision: 18 February 1997 / Accepted: 22 February 1997     

The relative arrangement of chromosomes in mitotic interphase and metaphase in Haplopappus gracilis

The relative position of mitotic metaphase chromosomes in Haplopappus gracilis is studied by direct observation of undisturbed metaphase cells in root tips: the homologous chromosomes lay always adjacent to each other, whereas the relative position of the pairs is not constant. — The relative position of interphase chromosomes is inferred from the frequency of radiation-induced mutual rearrangements between any possible pair of chromosomes. — It is concluded that the relative position of interphase chromosomes is reflected by the relative position of metaphase chromosomes in Haplopappus gracilis.     

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis

Summary Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10^-6M 2,4-D. Transfer of cells to media with 10^-5M kinetin or benzyladenine and no auxin or 10^-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10^-5M kinetin and with 10^-7 M NAA or 5×10^-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10^-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10^-7M but not at 10^-6M. At 10^-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.Issued as NRCC No. 11388.Abbreviations used: 2,4-D=2,4-dichlorophenoxyacetic acid, NAA + -naphthaleneacetic acid.     

Productivity of autotetraploids and frequency of aneuploidy in reproduction in Haplopappus gracilis L.]

Comparative studies of productivity of autotetraploid plants H. gracilis L. after selection for high productivity, without selection for high productivity and aneuploidity, and after selection for low productivity have been done. The results show considerable effectiveness of selection for productivity. Presence of hyperaneuploid forms in the population is a major cause of the decreasing of autotetraploid productivity.     

Efficient isolation of non-chimeric tetraploids artificially induced in a stable culture of Haplopappus gracilis

A method for reducing cytochimerism and inducing homogeneous tetraploids in Haplopappus gracilis (2n = 4) was developed in which masses of shoot primordia treated with 0.5 mg/ml of colcemid for 3 days were cut into small meristematic domes. All of the shoot primordia sampled just after the colcemid treatment were cytochimeras that were mixoploids of 2x, 4x and 8x cells. However, when they were allowed to recover in a colcemid-free medium, the frequency of 4x cells spontaneously increased in most of the shoot primordia. Thirty days after the recovery, chimeric masses containing shoot primordia, each of which consisted uniformly of 4x or 2x cells, were observed. In order to obtain a completely homogeneous tetraploid mass, we then cut these primordia into small pieces, each of which had approximately one meristematic dome. Subsequent to this homogeneous tetraploid masses were easily obtained. Tetraploid shoot primordia could propagate with chromosomal stability over a year, and plants regenerated from these tetraploid shoot primordia were also completely tetraploid. These results show that non-chrimeric masses can be easily isolated from artificially induced cytochimeras using masses of shoot primordia as material.