Increased ethylene and decreased phenolic compounds stimulate somatic embryo regeneration in leaf thin section cultures ofDoritaenopsis hybrid

The effects of endogenous levels of ethylene and phenolic compounds on somatic embryogenesis, medium-browning, and peroxidase activity were evaluated in thin section cultures ofDoritaenopsis. Cultures were maintained for 8 weeks with four different treatments: i) thick leaf segment culture, ii) thin leaf section culture, iii) thin leaf section culture with ventilation, or iv) thin leaf section culture after expiants were first washed. Expiants cultured in closed vessels produced a larger number of somatic embryos than those reared in the ventilated vessels. This enhanced formation confirmed the greater involvement of accumulated ethylene under non-ventilated conditions, because wound-induced tissues from thin leaf sections normally release high level of ethylene. When expiants were washed in the liquid medium and inoculated on the same solid medium, somatic embryo production was 1.7 and 18.5 times higher than in the thin section cultures and thick segment cultures, respectively. Reducing the level of phenolics in expiants at the initial stage of culturing apparently stimulated this embryo regeneration.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

Effect of N-Methyl-N-nitrosourea and N-Methyl-N-nitrosourethane upon the growth of tissue cultures ofNicotiana tabacum L.

N-Methyl-N-nitrosourea and N-methyl-N-nitrosourethane at concentrations of 0.1 mM to 1 mM inhibited the growth of tissue cultures ofNicotiana tabacum L. The inhibitory effect was proportional to the mutagen concentration applied. The primary expiants (pith slices) and a 3-year tissue culture strain exhibited a different sensitivity to the same mutagen concentrations. The variability in sensitivity of tissue culture inocula to mutagen effects was reduced by previous fractionation of the culture and by standardization of the age and size of inocula. The changes investigated in the ratio of relative growth rates between the controls and treated cultures give evidence of a fluctuating expression of mutagen effect in the course of the subculture interval and may demonstrate a recovery of the cultures from the mutagen effect.     

The study of morphological and histological changes in tissue cultures ofmatricaria inodora L

This paper deals with some morphological and histological changes observed during regular intervals inMatricaria inodora L. tissue cultures derived from leaf expiants. The expiants were cultivated on Murashige-Skoog’s culture medium supplemented with 1.0 mg 1⁻¹ 2,4-dichlorophenoxyacetic acid. The callus formation started about a week after isolation. During the second week the meristematic centers were differentiated from which root and shoot apices were later formed. During long term cultivation under the same culture conditions the inhibition of development of shoot apices took place. Only roots of unorganized growth have been regenerated. The influence of culture conditions on morphogenetic potential is discussed.     

The effect of irradiance and irradiation time on the size of initial cells in vegetative cell enlargement of Coscinodiscus wailesii (Centrales, Bacillariophyceae) in culture

The effect of irradiance and irradiation time were examined on rates of pseudo-auxospore formation and the size of initial cells in vegetative cell enlargement of the giant diatom Coscinodiscus wailesii Gran in culture. The mean rate of pseudo-auxospore formation ranged from 25.9% to 47.8% between experimental incubation conditions. No significant difference was detected in rates among different irradiances and irradiation times, and parent cell sizes. However, the mean valve diameter of the initial cells of the diatom was affected by an interaction between the light conditions and the diameter of the parent cells. Initial cells size tended to be larger with combined conditions of higher irradiance levels and longer irradiation time. There appears to be a relationship (a steady increase, although not a straight line) between the total daily irradiance and the valve diameter of the initial cells. The mean valve diameter of initial cells from larger parent cells was significantly larger than those from smaller parent cells under the same light conditions.     

Influence of Leaching Parameters on the Biological Removal of Uranium from Coal by a Filamentous Cyanobacterium

Axenic cultures of the filamentous cyanobacterium LPP OL3 were incubated with samples of uraniumbearing coal from a German mining area. The influence of leaching parameters such as coal concentration (pulp density), initial biomass, particle size, temperature, and composition of the growth medium on the leaching of uranium from the ore by the cyanobacterial strain was studied. When low pulp densities were applied, the yield of biologically extracted uranium was optimal (reaching 96% at 1% [wt/vol] coal) and all released uranium was found in the culture liquid. Above 10% (wt/vol) coal in the medium, the amount of cell-bound uranium increased. Initial biomass concentration (protein content of the cultures) and particle size were not critical parameters of leaching by LPP OL3. However, temperature and composition of the growth medium profoundly influenced the leaching of uranium and growth of the cyanobacterium. The yield of leached uranium (at 10% [wt/vol] coal) could not be raised with a tank leaching apparatus. Also, coal ashes were not suitable substrates for the leaching of uranium by LPP OL3. In conclusion, the reactions of the cyanobacterium to variations in leaching parameters were different from reactions of acidic leaching organisms.     

Plant regeneration from sugarbeet (Beta vulgaris L.) hypocotyls cultured in vitro and flow cytometric nuclear DNA analysis of regenerants

A simple and reproducible protocol for regeneration of sugarbeet plants from hypocotyl expiants derived from 21 day-old-seedlings has been developed. Expiants were cultured on MS medium containing 0.3 mg/l N6-Benzylaminopurine, 0.1 mg/l Naphthalene Acetic Acid, 50 mg/l adenine and 0.5% (w/v) fructose, 0.5% (w/v) sucrose and 0.5% (w/v) glucose to induce the formation of organogenic calli (2.3% to 46.5% organogenic efficiency, depending on populations). Shoot formation was induced in callus cultures of more than 1600 genotypes. Physiological age affected culture response and different genotypes had different temperature optima for organogenesis. Following transfer of regenerated plants to the greenhouse, DNA determinations were made to study the stability of ploidy. Differences in ploidy were observed in plants derived from both shortterm and long-term callus cultures; diploid true-to-type regenerants were 96% and 83%, respectively, from shortterm and long-term callus cultures.Abbreviations MS Murashige and Skoog medium - BAP N6-benzylaminopurine - IBA Indolebutyric acid - NAA Naphthalene acetic acid - TIBA 2,3,5 triiodobenzoic Acid - GM Germination Medium - IM Induction Medium - RG Regeneration Medium - RM Rooting Medium     

Influence des pretraitements par KCl et des rapports Ca/Na du milieu sur la croissance de vitroplants de tomate en milieu NaCl

The 18-day-old tomato vitroplants were obtained in axenic conditions by culture of expiants (including the terminal bud and the last internode of the stem) on agar-agar nutritive medium with 0 or 75 mM NaCl. The growth and the mineral content of the vitroplants were compared when the expiants were grown on media either with low or high K/Na and Ca/Na ratios, or with low K/Na and Ca/Na ratios after pretreatments of expiants by KC1, NaCl or CaCl₂ (from 0 to – 4.5 bar). The KCl pretreatment (-1.1 bar) during one day brings about an increase in vitroplant growth greater than that produced by a high Ca/Na ratio medium. The Cl accumulation was similar in expiants pretreated by KCl or NaCl. Ion content per gram of fresh matter was similar in 18-day-old vitroplants pretreated by KCl, NaCl or CaCl₂; the Na accumulation by KC1 pretreated vitroplants was not lower than that of 18-day-old vitroplants grown on a high Ca/Na ratio medium. These results show the relation between Na content of expiants and the growth of vitroplants in a NaCl medium.     

PATTERNS OF RELATIVE GROWTH IN NEREOCYSTIS LUETKEANA (PHAEOPHYTA)1

Plants of Nereocystis luetkeana (Mert.) Post. et Rupr. of different sizes were held on a raft at the surface of the sea off the Friday Harbor Laboratories, San Juan Island, Washington for 5 day periods for observation of detailed relative growth of different parts. Each stipe was marked at intervals with injected Indian ink and each blade was punched with a series of holes. Measurements of diameter, length, width and thickness were made before and after the 5 day periods. Blades showed a very similar pattern of relative growth rate (R) over an 18-fold range of sizes. The maximum local R in length was about 0.2 day⁻¹ and occurred at 6.5% of the distance from the bulb to the tip, declining to 0.01 at half way. Half the linear growth occurred in the proximal one tenth of the blade and 95% within the proximal half. The relative growth rate of the whole blade declined only slightly with increased size and lay between 0.0.3 and 0.06 day⁻¹ (approx. 3–6% day⁻¹). The linear growth rate therefore increased with blade size, the maximum observed being 14 cm day⁻¹. The maximum relative growth rate in blade width was slower, and sited more distally than that in length. Unless fertile tissue was involved all blade tissue, except that closely adjoining the bulb, became thinner during growth. R in volume reached 0.3 day⁻¹. Presumably because the plants were held near the sea surface stipes grew slowly, with a maximum linear rate of 9 mm day⁻¹. The maximum R in length decreased with stipe length. Bulb R in volume also decreased as size increased, from a maximum of 0.3 day⁻¹.     

PATTERNS OF RELATIVE GROWTH IN NEREOCYSTIS LUETKEANA (PHAEOPHYTA)1

Plants of Nereocystis luetkeana (Mert.) Post. et Rupr. of different sizes were held on a raft at the surface of the sea, off the Friday Harbor Laboratories, San Juan Island, Washington for 5-day periods for obseruation of detailed relative growth of different parts. Each stipe was marked at intenlals with injected Indian ink and each blade was punched with a series of holes. Measurements of diameter, length, width and thickness were made before and after the 5-day periods. Blades showed a very similar pattern of relative growth rate (R) ovler an 18-fold range of sizes. The maximum local R in length was about 0.2 day⁻¹ and occurred at 6.5% of the distance from the bulb to the tip, declining to 0.01 at half way. Half the linear growth occurred in the proximal one tenth of the blade and 95% within the proximal half: The relative growth rate of the whole blade declined only slightly with increased size and lay between 0.03 and 0.06 day⁻¹ (approx. 3-6% day⁻¹). The linear growth rate therefore increased with blade size, the maximum observed being 14 cm day⁻¹. The maximum relative growth rate in blade width was slower, and sited more distally than that in length. Unless fertile tissue was involved all blade tissue, except that closely adjoining the bulb, became thinner duringgrowth. R in volume reached 0.3 day⁻¹. Presumably because the plants were held near the sea surface stipes grew slowly, with a maximum linear rate of 9 mm day⁻¹. The maximum R in length decreased with stipe length. Bulb R in volume also decreased as size increased, from a maximum of 0.3 day⁻¹.     

苦瓜植株不同构件层次对支持物直径变化的行为反应

 采用实验生态学的方法,在提供不同直径支持物的情况下,对攀援植物苦瓜(Momordica charantia)不同构件层次的形态可塑性反应、生物量积累和生物量配置的变化进行了研究。结果表明：1)支持物直径对植株分枝出现的时机无明显影响,但分枝数量和分枝在主茎上的着生位置受支持物直径影响较大;2)植株个体水平比分枝水平对支持物直径的变化有更灵敏的反应,当支持物直径较大时,植株个体表现出对支持物较强的寻觅能力：主茎伸长受阻,比茎长变大,分枝数量和分枝率增大,分枝长度增加,比叶柄长度增加,根冠比减小;3)分枝形态特征和生物量特征对支持物直径的变化均无显著反应;4)苦瓜植株个体水平上的形态特征变化、生物量配置格局的改变以及分枝行为的变化是植株对支持物有效性变化的响应,有利于增强植株“寻觅”支持物的能力。     

pH and Glucose Profiles in Aggregates of Bacillus laevolacticus

Size distributions and glucose and pH profiles of aggregates of the d-(-)-lactic acid-producing organism Bacillus laevolacticus were measured. The organisms were grown in continuous culture with a medium glucose concentration of either 280 or 110 mM. A maximal aggregate diameter of 2.2 mm, with a Sauter mean of 1.46 mm, was determined for the former culture condition, whereas aggregates from a culture with 110 mM glucose input had a maximal diameter of 1.9 mm (Sauter mean of 1.07 mm). A pH gradient of approximately 2 U was observed for large aggregates (above 1.5 mm). In smaller aggregates (0.75 mm), the pH value in the interior part was approximately 0.4 U lower than that in the culture fluid. It could be concluded that, in cultures with the high glucose input, lactic acid accumulated within the aggregates to such an extent that metabolism in the central region of the larger aggregates could not proceed further. In these cultures, approximately 90% of the total biomass was active. In aggregates from cultures with a low glucose input, glucose only partly penetrated the larger-sized aggregates, and the activity of this culture was reduced to approximately 70% of the biomass. These aggregates were found to decrease in size after prolonged periods of cultivation. It is suggested that this is caused by glucose depletion in the interior of the aggregates. It is concluded that the availability of glucose is an important factor in determining the size of aggregates of B. laevolacticus.     

Effects of follicular fluids on the growth of porcine preantral follicle and oocyte

We examined the effect of supplementing the culture medium with follicular fluid (FF) on the growth of porcine preantral follicles and oocytes. Firstly, preantral follicles were retrieved from ovaries and then FF was collected from all antral follicles that were 2-7 mm in diameter (AFF), which included large follicles of 4-7 mm in diameter (LFF) and small follicles of 2-3 mm in diameter (SFF). When preantral follicles with a diameter of 250 mum were cultured in medium containing AFF, the growth of follicles and oocytes was greater than when follicles were cultured in medium containing fetal calf serum (FCS). When this growth-promoting effect in AFF was compared for LFF and SFF, the LFF were shown to be significantly more effective than SFF. This LFF effect was lost, however, when the concentration of LFF in the medium was decreased from 5% to 0.5% or when LFF were heat treated (60 degrees C for 30 min) or trypsin was added. In contrast, a decrease in SFF concentration from 5% to 0.5% and heat treatment of the SFF enhanced preantral follicle growth. Furthermore, proteins obtained from LFF that had molecular weights greater than 10 kDa (LFF > 10 kDa) had similar, but relatively reduced, growth-promoting properties. The remaining three LFF protein fractions (<10 kDa or <100 kDa or >100 kDa), however, did not have these growth-promoting properties. In conclusion, the supplementation of medium with LFF, rather than serum, enhanced preantral follicle and oocyte growth. Factors that enhanced follicle development in LFF and factors that suppressed follicle development in SFF were proteins and these LFF factors ranged in size from 10 kDa to over 100 kDa.     

A comparison of oxygenation methods fro high-density perfusion culture of animal cells

A perfusion culture system was developed to investigate the oxygenation of high-density hybridoma cell cultures. The culture system was composed of a stirred-tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble ( approximately 5 mm in diameter), micron-sized bubble ( approximately 80 mum in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron-sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble-free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high-density animal cell cultures, provided that the culture medium is supplemented with Pluronic F-68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long-term cultivation were found to be more problematic than simple bubble oxygenation of high-density cell culture. The micron-sized bubble oxygenation method is highly recommended for high-density animal cell cultures, provided that Pluronic F-68 is supplemented into the culture medium. (c) 1993 John Wiley & Sons, Inc.     

Studies on lavandin callus cultures: Ethylene production in relation to the growth

Ethylene production and growth of callus cultures of lavandin (Lavandula offidnalis Cham x Lavandula latifolia Villars) cv. Grosso were examined. Callus lines, derived from various kinds of primary expiants (shoot tip, leaf and calyx), exhibited differences in ethylene production patterns independent of callus growth. Moreover these differences could not be ascribed to the expiant source. Within a line, ethylene pattern paralleled callus growth curve. Variations in ethylene evolution were induced in shoot tip callus by means of ACC, AVG and varied amounts of 2,4-D in the culture medium. Following all these treatments callus growth was not altered. Hie decrease in 2,4-D concentration caused changes in Chl a and water content of the tissues.     

Regeneration responses of callus from different expiants and changes in isozymes during morphogenesis in wheat

Immature embryo and root meristem expiants of wheat were cultured on modified medium of Murashige and Skoog and Gamborg’s medium supplemented with 2,4-dichlorophenoxy acetic acid. Morphogeriic callus cultures were obtained from both the expiants. The frequency of shoot formation varied from 22% to 48% from callus obtained from embryos while only root formation could be induced from root meristem expiants. Cultures from young and old non-differentiating calli, and calli with shoot and/or root formation at different intervals were analysed for isozymes of esterase, peroxidase and acid phosphatase for studying the morphogenic capacity. With the development of shoot and/or root from callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root/shoot development rather than in the induction of morphogenesis in callus. Basic isozyme pattern of each enzyme for the callus was retained in all the callus stages.     

Cell Growth and Flavonoids Production in Suspension Culture of Saussurea medusa

Cell suspension cultures were established from Saussurea medusa Maxim. callus cultures. The effects of different rotation speeds of the gyratory shaker, different inoeulum sizes and different pH values of the medium on cell growth and flavonoid formation were studied. The result showed that the optimum rotation speed, inoeulum size and initial pH value of the medium were 90–120 r/min,50– 80 g FW/L and 5.5–6.0 respectively for cell growth and flavonoids formation in the suspension cultures. Sucrose was better than glucose and fructose for the suspension cultures. The optimum concentration of sucrose for cell growth and flavonoid production was 40 g/L, and the concentration of flavonoids could be as high as 1 423.25 mg/L. High performance liquid chromatographic analysis of cell suspension culture extracts showed that the concentrations of jaceosidin and hispidulin in the flavonoids were 22.11% and 0. 15% respectively.     

Cell-free plasma layer in cerebral microvessels.

Two diameters of vessel and red cell column in cerebral microvessels (> 29.8 microns in diameter) of cat were measured together with red cell velocity, using a two fluorescent tracer method. A fluorescein isothiocyanate (FITC)-labeled red cell was adopted as a flow tracer to measure the cell velocity with a dual window technique. Based on the fluorescence image, the red cell column diameter was measured. Plasma was stained with rhodamine-B isothiocyanate (RITC)-labeled dextran to measure the vessel diameter. The thickness of the cell-free plasma layer could be determined from the difference of the two diameters. The obtained thickness of the cell-free layer was not described by a simple function of vessel diameter or red cell velocity; it was dependent on the pseudo shear rate defined by the ratio of cell velocity to vessel radius. The layer thickness increased with a decrease in the pseudo shear rate.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.