Carbohydrate,metabolic, and osmotic changes in scaled-up liquid cultures of <Emphasis Type="Italic">Narcissus</Emphasis> leaves

Summary The use of scaled-up liquid cultures could be an efficient system for mass propagation of Narcissus, as it can greatly reduce the costs involved with manual handling. Induction of hyperhydric meristematic leaf section clusters and proliferation were carried out in an ancymidol (ANC)-containing liquid medium in flasks and disposable presterilized plastic bioreactors. Non-hyperhydric bulblets started to develop from hyperhydric meristematic leaf section clusters after subculture on a 0.8% agar strength medium, and young bulbs formed after 10 mo. in vivo acclimatization with a 98% survival rate. The present study reveals that in Narcissus leaf sections cultured in liquid medium, morphogenetic changes in leaf sections were associated with metabolic changes. The changes in carbohydrate, protein, and water potential of the liquid media and leaf sections were found to be closely related to meristematic center initiation on Narcissus hyperhydric leaf sections. Starch, sucrose, and glucose were significantly higher in the hyperhydric leaf sections cultured in ANC medium. The water potential was signifieantly higher in ANC-treated leaf sections and significantly lower in the medium containing ANC, at the stage shortly before or after hyperhydricity and meristematic centers hegan forming on the leaf sections. A 30kDa protein was found to be present in the hyperhydric leaf sections. Based on the present study, a largescale micropropagation protocol of Narcissus in agar and liquid cultures is proposed.     

Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling: implications of oxidative stress

Carnation shoot cultures were micropropagated in two different agar concentrations (0.58 and 0.85%) and placed in a bottom cooling system or control conditions. During the culture period of 28 days, it was observed that relative humidity, hyperhydricity, dry weight, multiplication rate, and the activity of the antioxidant enzymatic system changed in relation to the agar concentration used and the application of bottom cooling. The percentage of hyperhydric shoots also showed a significant decrease under bottom cooling conditions for both agar concentrations. Lipid peroxidation was always lower in shoots cultured with bottom cooling. All the antioxidant enzymatic activities were lower in bottom cooling treatments compared to controls. These results show that the normalization of the environmental conditions in vitro via bottom cooling can prevent the onset of different simultaneous stress reactions concomitant with hyperhydricity. The present work provides for the first time , direct evidence of a reduced H₂O₂ generation in the tissues cultured in bottom cooling able to reduce oxidative stress.     

The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus

 Addition of the growth retardant ancymidol to Narcissus shoots and lower inner leaf sections isolated from shoots cultured in liquid medium induced hyperhydric malformations associated with morphogenetic changes. Meristematic centers initiated on the basal proximal ends appeared over the entire surface of the hyperhydric leaf sections after 6 weeks in culture. The meristematic centers which formed clusters on the leaf sections developed later into buds. In leaf sections grown in the liquid medium lacking ancymidol, hyperhydricity was not induced, and regeneration was not observed. Starch and protein levels and ascorbate peroxidase and catalase activities were examined in shoots and isolated leaf sections that were either hyperhydric or non-hyperhydric. In ancymidol-treated, hyperhydric leaf sections, ascorbate peroxidase and catalase activities were lower than in control, untreated leaf sections. The changes in starch and protein levels and in antioxidant enzymatic activities appeared to be related to the onset of meristematic-center initiation and further bud development on Narcissus hyperhydric leaf sections. Received: 6 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Propagation and tuberization of potato bud clusters from bioreactor culture

Summary Single node stem segments fromin vitro potato shoots cultured in liquid medium in the presence of ancymidol (23.4 μM) developed into bud clusters in either shaken flasks or bioreactor cultures. Buds on the clusters developed tubers after subculture to a tuber induction medium with 23.2 μM kinetin, 19.5 μM ancymidol, and 6-8% sucrose. The number of tubers per cluster and their size were higher in agar induction medium on top of which a second layer of liquid medium was added, than in liquid shake or bioreactor cultures. The highest increase in tuber size (i.e., 720 mg fresh weight after 7 weeks), was obtained in agar cultures flushed twice with liquid tuber induction medium. The potential of bioreactor cultures for potato bud proliferation and enhanced tuber development in double layer agar-liquid cultures is discussed.     

Ancymidol-enhanced hyperhydric malformation in relation to gibberellin and oxidative stress in liquid-cultured <Emphasis Type="Italic">Narcissus</Emphasis> leaves

Summary The growth retardant ancymidol inhibited gibberellin biosynthesis and enhanced hyperhydric malformation of Narcissus leaf sections cultured in liquid medium. Superoxide dismutase activities were examined by spectrophotometry and native polyacrylamide gel analysis, and gibberellin and hydrogen peroxide levels were determined spectrophotometrically in either hyperhydric or non-hyperhydric leaf sections. In ancymidol-treated hyperhydric leaf sections, superoxide dismutase activity and hydrogen peroxide levels were higher during the initial culture period, when hyperhydric malformation occurred, than in control untreated leaf sections. At a later stage, when the meristematic centers started to form on ancymidol-enhanced hyperhydric leaf sections, superoxide dismutase activity, hydrogen peroxide, and gibberellin levels were significantly lower in hyperhydric leaf sections than in non-treated leaf sections. The changes in superoxide dismutase activities, hydrogen peroxide, and gibberellin levels appeared to be related to hyperhydric malformation and meristematic center initiation.     

Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures

In vitro shoot regeneration of gladiolus in three different culture systems, viz., semi-solid agar (AS), membrane raft (MR), and duroplast foam liquid (DF) cultures was evaluated following the kinetics of shoot multiplication and hyperhydricity at optimized growth regulator combinations. Compared to the AS system, matrix-supported liquid cultures enhanced shoot multiplication. The peak of shoot multiplication rate was attained at 18 days of incubation in the MR and DF systems, whereas the maximum rate in the AS system was attained at 21 days. An early decline in acceleration trend was observed in liquid cultures than the AS culture. The hyperhydric status of the regenerated shoots in the different culture systems was assessed in terms of stomatal attributes and antioxidative status. Stomatal behavior appeared to be normal in the AS and MR systems. However, structural anomaly of stomata such as large, round shaped guard cells with damage in bordering regions of stomatal pores was pronounced in the DF system along with a relatively higher K⁺ ion concentration than in the AS and MR systems. Antioxidative status of regenerated shoots was comparable in the AS and MR systems, while a higher incidence of oxidative damages of lipid membrane as evidenced from malondialdehyde and ascorbate content was observed in the DF system. Higher oxidative stress in the DF system was also apparent by elevated activities of superoxide dismutase, ascorbate peroxidase, and catalase. Among the three culture systems, liquid culture with MR resulted in maximum shoot multiplication with little or no symptoms of hyperhydricity. Shoots in the DF system were more prone to hyperhydricity than those in the AS and MR systems. The use of matrix support such as membrane raft as an interface between liquid medium and propagating tissue could be an effective means for rapid and efficient mass propagation with little or no symptoms of hyperhydricity.     

Improved in vitro rooting and hyperhydricity in regenerating tissues of Thapsia garganica L.

Stock cultures of Thapsia garganica grown on Murashige and Skoog agar medium (1962) (MS) (0.8% agar [w/v]; pH 5.8) with 0.5 mg l⁻¹ NAA and 1.5 mg l⁻¹ BA were best rooted by subjecting to half strength MS liquid medium with IBA (10 mg l⁻¹) for 3 days prior to transfer to medium without plant growth regulators. A rooting frequency of 60% was noted with seven roots per rooted plant. Rooting was apparent after 10 days. The present study also aimed at reducing the occurrence of hyperhydric plants. The inclusion of 2% polyethylene glycol (w/v) in the growth medium or ventilation of cultures prior to acclimatization resulted in the production of plants true to type, closely resembling wild plants. Those plantlets that had been rooted and exposed to a better vented environment were able to acclimatize readily. Tissue culture propagation is therefore beneficial to the conservation of the medicinally important species, Thapsia garganica.     

Enhanced embryogenesis and plant regeneration from cucumber (Cucumis sativus L.) callus by activated charcoal in solid/liquid double-layer cultures

Enhanced embryogenesis and plant regeneration methods were established in cucumber (Cucumis sativus L. cv. ‘Delilah’) using hypocotyl segments as explants. Callus formation, followed by pro-embryogenic aggregates and globular embryoids required liquid shake cultures. In liquid medium, however, many of the embryoids developed into abnormal structures — ‘neomorphs’ or succulent plantlets. Embryoids subcultured to stationary liquid or agar cultures dedifferentiated and underwent secondary embryogenesis. Neither increased osmolarity nor adding abscisic acid (ABA), zeatin or activated charcoal to the liquid medium inhibited abnormal morphogenesis. The use of double layer cultures containing activated charcoal in the lower agar layer and ABA with elevated calcium in the upper liquid phase prevented dedifferentiation and secondary embryogenesis and allowed normal organized growth of the embryoids. Hardening in vitro by partial desiccation with CaSO₄ under aseptic conditions improved the cucumber plantlet's leaf growth and their survival after transplanting to soil.     

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus L

Summary Comparative studies of carnation micropropagation under four different ventilation rates showed that using gas-permeable filters, with gelled or liquid media and modifying the volume of culture medium, it was possible to establish a suitable hydric state to obtain good proliferation rates with gelled and liquid medium, as well as optimal acclimatization of microcuttings. The following parameters were measured: ventilation rate, gas exchange coefficients, relative water loss, increase of agar concentration, micropropagation rates, percentage of hyperhydricity, and acclimatization rates. Our results confirm that it is possible to avoid hyperhydric plants cultured in liquid medium with the use of ventilated culture vessels through the control of the water relations during the multiplication phase and, at the same time, keeping the micropropagation rate.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

Effects of irradiated medium on chromatid aberrations in mammalian cells using double mylar dishes.

We examined the potential contribution of irradiated medium on the bystander effect using custom made double-mylar stainless steel rings. Exponentially growing Human-hamster hybrid (AL) cells were plated on either one or both sides of double-mylar dishes 2-4 days before irradiation. One side (with or without cells) was irradiated with alpha particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facilities of Columbia University. Since alpha particles can only traverse a very limited distance, cells plated on the other side of a medium-filled mylar dish will not be irradiated by the alpha particles. The results of chromosomal aberrations on un-irradiated target cells that were attached to the top mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium filled chambers than just medium alone. Furthermore, when transferring the medium from these cell-irradiated dishes to fresh AL cultures, chromatid-type aberrations were produced in the un-irradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain modulating factors secreted from the irradiated cells on the bottom mylar layer into the medium, induce chromatin damage in the un-irradiated, bystander cells.     

Zeatin and TDZ-induced Shoot Proliferation and Use of Bioreactor in Clonal Propagation of Medicinal Herb, Roseroot (Rhodiola rosea L)

A procedure for in vitro propagation of roseroots (Rhodiola rosea L), a medicinal plant, was developed using a RITA bioreactor system containing liquid medium, combined with a gelled medium. Wild roseroot clones: ‘RCi’, ‘RC2’ and ‘RC3’ were established on a basal medium (BM) from in vitro-germinated seedlings on half-strength Murashige and Skoog (MS) salts. TDZ at 2–4 μM supported shoot proliferation but inhibited shoot elongation of ‘RCi’ shoots on gelled medium. Clones differed significantly with respect to multiplication rate with ‘RCi’ producing the most shoots per explant on gelled BM with 2 μM zeatin. In a bioreactor system, TDZ supported rapid shoot proliferation at lower concentration (0.5 μM) but induced hyperhydricity at more than 0.5 μM. Bioreactor-multiplied hyperhydric shoots of all clones when transferred to gelled medium containing 1–2 μM zeatin produced normal shoots within 4 wk of culture. Shoots were rooted in vitro on BM void of growth regulators. Almost all (9U to 95%) in vitro plantlets survived when transferred to potting medium.     

Influence of ambient air temperature on the cooling/heating load of a single cell protein jacketed fermenter operating on cheese whey under continuous conditions

The heat generated by mixing and lactose metabolism, during the continuous production of single cell protein from cheese whey lactose using a jacketed fermenter with running cooling water, was calculated using a heat balance equation. The technique quantified the heat produced in and lost from the fermentation unit. Most of the heat generated by mixing in the cell-free system (97.47%) was lost with exhaust gas, while a very small amount (2.53%) was lost through the fermenter lid, wall, and bottom. The heat generated by mixing was significant (26.31% of the total heat generated in the fermentation system with an active yeast population present) and, therefore, cannot be ignored in heat balance calculations. About 19.71% of the total heat generated in the reactor was lost through the coolant at an ambient temperature of 22 +/- 0.5 degrees C, showing the need for a cooling system. A yeast population size of 986 million cells/mL and a lactose removal efficiency of 95.6% were observed. About 72.5% and 27.5% of the lactose consumed were used for growth and respiration, respectively. A yield of 0.66 g of cells/g of lactose was achieved. The heat released by unit biomass was 7.05 kJ/g of cells. The results showed the significant impact of ambient air temperature on the cooling load. The heat to be removed from the medium by the cooling system varied from 3.46 to 281.56 kJ/h when the temperature increased from 16 to 30 degrees C. A heating system is needed to maintain the medium temperature at 34 degrees C when the ambient air temperature is below 16 degrees C.     

Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress

The physiology of hyperhydricity in relation to oxidative stress, mineral nutrients, antioxidant enzymes and ethylene has been studied in three micropropagated carnation cultivars under experimentally induced hyperhydricity. A marked increase in Fe content in comparison with normal tissues was observed in the hyperhydric tissues from the three cultivars. The levels of ethylene, solute leakage and malondialdehyde content were also significantly higher in the hyperhydric tissues. In relation to the time course of H₂O₂ production measured by fluorescence quenching, a similar trend could be observed for the three cultivars, with a clear increase in the generation of hydrogen peroxide in hyperhydric tissues. The activities of all the antioxidative enzymes studied, except lipoxygenase, were higher in the hyperhydric shoots. Phenylalanine ammonia-lyase (PAL) showed a significant decrease in activity in the hyperhydric tissues in comparison with the controls for the three cultivars. Soluble guaiacol peroxidase had a strong increase in activity in hyperhydric shoots of the three cultivars. These results provide, for the first time, direct evidence of H₂O₂ generation in hyperhydric tissues, characterize the response of the antioxidant system to an oxidative stress during hyperhydricity in carnation leaves and point to the accumulation of toxic forms of oxygen as the inducer of some of the abnormalities observed.     

Effect of medium on chromatin damage in bystander mammalian cells

In the present study, we examined the potential contribution of irradiated medium to the bystander effect using custom-made double-Mylar stainless steel rings. Exponentially growing human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-Mylar dishes 2-4 days before irradiation. One side (with or without cells) was irradiated with alpha particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facility of Columbia University. Since alpha particles can traverse only a very limited distance (around 23 microm in water), cells plated on the other side of a medium-filled Mylar dish will not be irradiated by the alpha particles. The results of the cytogenetic assay of unirradiated target cells that were attached to the top Mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium-filled chambers than with just medium alone. Furthermore, when the medium was transferred from these cell-irradiated dishes to fresh A(L) cell cultures, chromatid-type aberrations were produced in the unirradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain unidentified modulating factors secreted from the irradiated cells on the bottom Mylar layer into the medium induce chromatin damage in the unirradiated bystander cells.     

Theoretical analysis of specimen cooling rate during impact freezing and liquid-jet freezing of freeze-etch specimens.

We have carried out a theoretical analysis of specimen cooling rate under ideal conditions during impact freezing and liquid-jet freezing. The analysis shows that use of liquid helium instead of liquid nitrogen as cooling medium during impact freezing results in an increase in a specimen cooling rate of no more than 30-40%. We have further shown that when both impact freezing and liquid-jet freezing are conducted at liquid nitrogen temperature, the two methods give approximately the same specimen cooling rate under ideal conditions except for a thin outer layer of the specimen. In this region impact freezing yields the highest cooling rate.     

An improved system for the <Emphasis Type="Italic">in vitro</Emphasis> regeneration of <Emphasis Type="Italic">Thapsia garganica</Emphasis> via direct organogenesis – influence of auxins and cytokinins

An improved protocol for mass multiplication directly from leaf material of Thapsia garganicawas developed. Using factorial experimentation, auxins (NAA, IAA, 2,4-D) and cytokinin (BA, kinetin) combinations at 0–2 mg l⁻¹ added to Murashige and Skoog (MS) medium with 30 g l⁻¹ sucrose and 8 g l⁻¹ agar (pH 5.8) were tested for their effect on direct regeneration on leaflet and petiole explants. Of the media tested, the 0.5:1.5 NAA:BA medium was comparable for direct shoot organogenesis to the 2 mg l⁻¹ kinetin supplemented medium. However, when shoots were multiplied on these media, the 2 mg l⁻¹ kinetin without auxins was most effective as it kept the percentage of callus-derived plantlets to 3% and the number of hyperhydric shoots were minimal at a frequency of 2% compared to 25% on the 0.5:1.5 NAA:BA medium. The 2 mg l⁻¹ kinetin medium induced adventitious bud formation in 36% of the explants after 30 days. When the cultures were transferred to the same medium for multiplication, an average of six shoots (4.3 cm) were derived from each shoot base. Other combinations resulted in callus formation that either preceded shoot production or occurred together with adventitious shoot induction; whereas the 2 mg l⁻¹ medium resulted solely in adventitious buds that readily converted and elongated to shoots. On the 0.5:1.5 NAA:BA medium which tended to induce hyperhydric shoots in culture, agar (0.8, [w/v]) (15% hyperhydric plantlets) was more useful in maintaining a high health status in regenerating plantlets than gellan gum (Gelrite^®; 0.25, [w/v]) (60% hyperhydric plantlets). Although rooting in vitro was difficult, 58% of the propagules were successfully acclimatized when plants were exposed to fungicidal solutions as pre- and post-acclimatization treatments. A comprehensive protocol that allows for a reduction in mortality due to damping-off diseases during ex vitro transplantation of the in vitro-derived T. garganica plantlets is reported. The acclimatization procedure presented here is potentially suited to other umbelliferous species where fungal rots hamper ex vitro establishment.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

Effect of sucrose on growth and galanthamine production in shoot-clump cultures of Narcissus confusus in liquid-shake medium

In order to produce galanthamine, an alkaloid currently being tested in Alzheimer's disease therapy, we have used in vitro organ cultures of Narcissus confusus (Amaryllidaceae) plants starting from two different explants: double scale segments with basal plate from bulbs (organogenic cultures), and mature seeds (callogenic-organogenic cultures). Shoot-clumps were induced from buds obtained from twin-scales and from organogenic calluses on a MS medium supplemented with 1 mg l⁻¹ 2,4-D and 5 mg l⁻¹ BA. Shoot-clumps were then developed partially submerged in a liquid medium. After one month of precondition, the shoot-clumps were cultured in liquid media with different concentrations of sucrose, from 3% to 18% (w/v) for 14 days. The growth of the regenerated plants treated with 9% sucrose was significantly greater. Under a photoperiod 16 h light/8 h dark, the shoot-clump cultures subjected to the two highest sucrose concentrations gave rise to higher dry weight/fresh weight ratios. Different doses of sucrose affected not only the alkaloid profile in the shoot-clump tissues but also that excreted to the medium. In all cases, shoot cultures of N. confusus were capable of galanthamine biosynthesis, with the best results at 9% sucrose concentration. This revised version was published online in June 2006 with corrections to the Cover Date.