Softening response of 1-methylcyclopropene-treated banana fruit to high oxygen atmospheres

Exposure to high O₂ concentrations may stimulate, have no effect or retard fruit ripening depending upon the commodity, O₂ concentration and storage time among other variables. The ethylene-binding inhibitor 1-methylcyclopropene (1-MCP) was used to investigate ethylene-mediated softening responses of Williams banana fruit exposed to elevated O₂ for various periods of time. Fruit softening was measured at 25 °C and 90% relative humidity. Exposure to high O₂ concentrations for 5 days resulted in accelerated softening. Softening of fruit treated with 1-MCP for 12 h followed by 5 days of storage in high O₂ atmospheres at 25 °C was enhanced with increasing O₂ concentration between 21 and 100%. However, overall softening was much less compared to non-1-MCP-treated fruit. Softening of 1-MCP-treated fruit was progressively enhanced with increasing holding time from 5 to 20 days. Fruit treated with 1-MCP and then held for 10 days in high O₂ atmospheres followed by exposure to ethylene for 24 h and subsequent storage for 5 days at 25 °C softened more rapidly than those held in air for 10 days. 1-MCP-treated fruit held in various high O₂ atmospheres can regain gradually the sensitivity to ethylene and finally ripen over time. Enhanced softening of fruit exposed to elevated O₂ concentrations suggests that high O₂ treatments enhance synthesis of new ethylene binding sites.     

Protoplast culture and plant regeneration ofPinellia ternata

A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 10⁵ protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1–2 mm in diameter) formed after 30–40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1^–1) and NAA (0.2 mg 1)^–1) could regenerate plants after 40–50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - KT kinetin - CH casein hydrolysate     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Responses of Drosophila melanogaster to atypical oxygen atmospheres

We examined physiological phenotypes of Drosophila melanogaster in hypoxic to hyperoxic atmospheres. We performed measurements on life span or behavioural function in 5, 21, 40, 60, and 80% O₂, and combined this with literature data for 2% and 100% O₂. O₂ incubation resulted in a concentration-dependent reduction of life span in both hypoxia and hyperoxia, though different measures of life span were affected differently. We also examined how behavioural and metabolic functions were affected by exposure to hyperoxia (up to 60% O₂). Climbing behaviour was measured as a fast (4 s) and slow (55 s) response in a negative geotaxis assay. In normoxia, both measures of climbing response declined exponentially until disappearing completely. Interestingly, survivorship was very high until the loss of climbing ability, after which it dropped rapidly. This pattern appeared accelerated in 40% O₂. However, while flies in 60% O₂ also apparently lost their fast climbing ability immediately prior to the drop in survivorship, they maintained considerable climbing ability over the longer trial. Metabolism, measured by CO₂ release, did not change with age in normoxic flies, but was significantly lower in flies exposed to hyperoxia, particularly as the flies aged. There was, however, a slight increase in water loss rate with age in normoxia, while in hyperoxia, water loss was reduced. Uniquely, the water loss rates of flies in 60% O₂ doubled immediately prior to the end of their life span. Because ageing results in generally irreversible functional declines, we examined if functional declines in hyperoxia (60% O₂) were also irreversible, or whether some functioning could recover after a return to normoxia. After 7 days of recovery, water loss rates decreased, CO₂ exhalation slightly increased, and climbing ability was partially recovered. Therefore, the effect of O₂ on D. melanogaster function is non-linear, may be reversible, and may include unique phenotypes that arise at some O₂ concentrations, and not others.     

Protoplast culture and plant regeneration in Lycium barbarum L.

Protoplasts were isolated from leaves of the woody plant Lycium barbarum L. and cultured in liquid nutrient medium TM-2 at a density of 10⁴–10⁵ cells ml^-1. After ten days of culture, regenerated colonies were transferred to the agar-solidified medium TM-3, and 5–7 days later to regeneration media PRM or TM-4. Formation of shoots was observed after 30–40 days. Completely formed and rooted plants were transferred to the soil. Cytological and morphological analysis of the regenerated plants revealed relative genetic stability of this species in the process protoplast — plant. The results obtained allow us to conclude that L. barbarum can be used in the experiments on somatic hybridization or direct gene transfer.     

Protoplast culture and plant regeneration from Brassica carinata Braun

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4–6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.Abbreviations BA 6-Benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashiqe-Skoog     

Protoplast culture of several members of the genus Physalis

High yields of protoplasts were obtained by enzymic treatment of mesophyll from five different species of the genus Physalis. Subsequent divisions and colony formation were achieved in all the species. However, numerous combinations of phytohormones failed to induce regeneration of shoots from callus tissue developed from protoplasts.     

Hyperbaric pressure of 51 atmospheres is without effect on the depression of oxygen uptake in kidney tissue culture produced by halothane

Although anesthetized animals are awakened when subjected to increased pressure, compression does not result in antagonism of all phenomena associated with these drugs. It has recently been demonstrated that halothane's inhibition of respiration of isolated rat liver mitochondria is not reversed by hydraulic compression to 51 atmospheres. In order to determine whether this phenomenon can be extrapolated to the whole cell, we have investigated the effect of hydraulic compression of intact renal cells equilibrated with halothane, and conclude that pressure does not overcome the inhibitory effect of this anesthetic.     

Protoplast culture and female plant regeneration in the dioecious plant Melandrium album

Conditions for protoplast isolation and culture in the dioecious species Melandrium album (Silene alba) are described and differences between female and male plants subjected to in vitro culture identified. Plant regeneration was achieved in the case of female plants only. Selection for high regeneration capacity lines was initiated aiming at creating responsive experimental material for further studies. The regeneration behaviour in this dioecious plant is discussed in the context of hormonal models on sex determination.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - 2iP N⁶-[2-isopentenyl]adenine - NAA -naphthalene-acetic acid - PVP polyvinylpyrrolidone     

Protoplast culture and somaclonal variability of species of series Juglandifolia

Mesophyll protoplasts of species of series Juglandifolia (Solanum rickii, S. lycopersicoides, S. ochranthum and S. juglandifolium) were isolated and cultured in liquid nutrient media TM-2 or KM8P. The cell colonies formed were transferred onto agar-solidified media TM-3 or GM, and 10 to 15 days later onto TM-4, PRM, MS3ZG, KK or C regeneration media. Formation of the shoots for S. rickii and S. lycopersicoides was observed after 30 to 35 days on regeneration medium. The regenerated shoots were rooted on hormone-free MS medium. Morphological and cytogenetic analyses have shown that somaclonal variants might arise in the course of plant regeneration from protoplasts of these species.Abbreviations BAP 6-benzylaminopurine - 2, 4-D 2, 4 dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - B5 Gamborg medium - TRIS tris(hydroxymethyl)aminomethane     

Protoplast culture and plant regeneration of several species in the genus Dianthus

Summary Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.Abbreviations MS Murashige and Skoog (1962) - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - FDA fluoroscein diacetate - NAA 1-naphthaleneacetic acid     

Protoplast culture and plant regeneration in Glycine canescens F.J. Herm

Protoplasts were isolated from seedling hypocotyls of Glycine canescens F.J. Herm. and cultured in agarose to form multicellular colonies. Colonies transferred to agar medium enlarged to form calli which were tested for morphogenic potential on a range of media. Protoplast-derived calli produced shoots with a low but reproducible frequency; these shoots were rooted and transferred to the greenhouse.     

High cell density perfusion culture of hybridoma cells recycling high molecular weight components

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.     

Development of one-cell ovine embryos in two culture media under two gas atmospheres

The objective of this study was to develop a successful system for culturing one-cell ovine embryos through several cleavage divisions. One hundred and four one-cell embryos were collected from synchronized, FSH-treated ewes 48 hr after the onset of estrus and randomly placed in one of four culture treatments. The effect of glucose supplementation and reduced oxygen tension (20% vs. 5%) on embryo development was studied. Embryo development was quantitated by a cleavage index based on the number of completed cell divisions. The number of embryos completing at least two cell divisions when cultured in Brinster's Pyruvate Medium (BPM) was 7 26 and 9 26 , under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while 22 26 and 20 26 embryos divided when cultured in BPM supplemented with 0.1% glucose (BPM-G) under similar atmospheres. Mean cleavage indices for embryos cultured in BPM were 1.2 and 1.6 under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while embryos cultured in BPM-G had mean cleavage indices of 4.6 and 4.0, respectively. Results of this study indicate that one-cell ovine embryos can be successfully cultured through several cleavage divisions. Glucose supplementation was beneficial for one-cell ovine embryo development. Reducing the oxygen tension from 20 to 5% had no effect on embryo development, and there was no media x gaseous atmosphere interaction.     

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree of inhibition of the demethylation reactions by a variety of CO:O₂ mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O₂ utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.     

Protoplast culture and transformation studies of Triticale (x Triticosecale Wittmack)

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Biphasic culture of rat lung slices for pharmacotoxicological evaluation of complex atmospheres

The relevance to the in vivo situation of in vitro toxicity studies of complex atmospheres has frequently been limited by the procedures used for the exposure of the biological samples. We have evaluated from on-road measurements the size distribution pattern and the subsequent respiratory tract deposition rates of particulate matter from urban atmospheric aerosols, which are in the range of 110 and 3 pg/cm² per min for tracheobronchial and alveolar areas, respectively. Continuous flow-through rotating chambers and a specific design for exhaust sampling and dilution with controlled adjustment of pO₂ and pCO₂ to 20% and 5%, respectively, have been developed to expose biphasic air/liquid organotypic cultures of rat lung slices to continuous flows of diluted exhausts from diesel engines with preservation of the physicochemical properties of the exhaust. The size distribution of the particulate matter and the bioavailability of pollutants were preserved, thus allowing us to closely mimic in vitro the in vivo atmosphere/tissue interactions that occur mainly through diffusion mechanisms. The toxicity response profile has been assessed in terms of tissue viability, oxidative stress, DNA injury, and the early phase of inflammatory reaction. Exhaust filtration, addition to fuel of rapeseed methyl ester, and preincubation of lung tissue with soy isoflavones modulated the toxicity response profile of exhausts. The importance of preserving both particulate matter size distribution and adsorbed pollutant bioavailability, which could not be ascertained using more classical in vitro approaches, is discussed and should be considered a prerequisite for further developments of in vitro studies to modelize in vivo inhalation of complex atmospheres. This revised version was published online in August 2006 with corrections to the Cover Date.