Effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis

The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving. Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1% activated charcoal, added before autoclaving. In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolyzed.     

Fermentation pattern of Zymomonas mobilis at high sucrose concentrations

Summary Zymomonas mobilis was grown in batch concentrations between 200 and 400 g/l sucrose. The fermentation pattern revealed that the efficiency of sucrose hydrolysis dropped only from 94 to 78.6% whereas the efficiency with which the hydrolyzed products were converted to ethanol decreased from 94 to 43%. The ethanol yields were relatively constant for final concentrations which lay between 80 and 132 g/l. Fermentation times increased to 72 hours at the higher sucrose concentrations. Sorbitol and fructose were identified as the major by-products. Preliminary evidence suggests that the ratio between the two by-products depends on the pH of the culture medium. Results suggest the possibility of processes producing ethanol plus fructose, ethanol plus fructose and sorbitol, or ethanol plus sorbitol in a single-stage batch fermentation.     

Sucrose uptake and metabolism in a double layer system for micropropagation ofRosa multiflora

Uptake and metabolism of sucrose in micropropagatedRosa multiflora using the double layer technique was investigated. In the multiplication as well as the root induction stage, hydrolysis of sucrose in the culture medium was observed. A mathematical model was developed to quantify sucrose hydrolysis and the uptake of sucrose, glucose and fructose, based on the time series for the different sugars in the culture medium. These data were linked to a study of the sugar metabolism in the microshoots. After 48 h of incubation on¹⁴C-[U]-glucose containing medium, the incorporated label was mainly detected in the ethanol soluble fraction; within this fraction sucrose was the most important compound. This indicates a significant re-synthesis of sucrose in the plant material after the uptake of hexose. To assess the extent that different enzymes of sucrose metabolism (invertases, sucrose synthase and sucrose-P-synthase) were involved, their activity in different plant parts (of final stage III microshoots) were assayed. A decreasing gradient for sucrose metabolising enzymes from the roots toward the leaves gave a good indication of how the different tissues depend on sucrose absorbed from the medium.     

Anther culture of Solanum genotypes

Anther culture response was examined in five Solanum genotypes. Large genotypic differences exist in response to culture and liquid culture media were found to be superior to agar solidified media systems. Pre-treatment effects on embryoid induction were also investigated and two of the genotypes displayed differential response to temperature stress pretreatment. In general the beneficial effect of charcoal in the media was confirmed. Successful embryoid production and plantlet regeneration was obtained from the wild potato species, Solanum papita. The influence of sucrose concentration on embryoid production was also investigated. Large genotype by sucrose interactions were detected and this was mainly due to the differential response of the tuberosum clones to increasing sucrose concentration when compared with S. papita. Embryoids were produced from this species in media containing 15% sucrose although the optimum concentration of sucrose for embryoid production was 9%. The possible role of anther culture techniques in gene introgression and potato improvement is discussed.     

Production of carotenoids by β-ionone-resistant mutant of <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> using various carbon sources

Batch culture kinetics of the red yeast, Xanthophyllomyces dendrorhous SKKU 0107, revealed reduction in biomass with glucose and lower intracellular carotenoid content with fructose. Figures were different when compared to sucrose, which is a disaccharide of glucose and fructose. In contrast, specific growth rate constant stayed between 0.094~0.098 h⁻¹, irrespective of the carbon sources employed. Although the uptake rate of glucose was found to be 2.9-fold faster than that of fructose, sucrose was found to be a more suitable carbon source for the production of carotenoids by the studied strain. When sugar cane molasses was used, both the specific growth rate constant and the intracellular carotenoid content decreased by 27 and 17%, respectively. Compared with the batch culture using 28 g/L sugar cane molasses, fed-batch culture with the same strain resulted in a 1.45-fold higher cell yield together with a similar level of carotenoid content in X. dendrorhous SKKU 0107.     

Maturation of black spruce somatic embryos: Sucrose hydrolysis and resulting osmotic pressure of the medium

The physiological and osmotic roles of sucrose during black spruce (Picea mariana (Mill.) B.S.P.) embryo maturation were investigated. The results showed that when both sucrose and mannitol were present in the medium, the optimum sucrose concentration varied between 4% and 6%. From these data, mannitol does not apparently replace sucrose during the maturation of somatic embryos and therefore it might not be a suitable osmoticum. For the media supplemented with 4% to 12% sucrose and various concentrations of mannitol, the osmotic pressure of the medium rose during maturation, particularly for the highest sucrose concentrations (7% to 12%). Medium containing 3% each of fructose and glucose produced fewer mature embryos compared to the medium with 6% sucrose. An increment in the osmotic potential was observed in medium with 6% sucrose in contrast to that containing 3% each of fructose and glucose. Sugar analysis revealed that the sucrose hydrolysis in the medium was detectable within 1 week of incubation and continued throughout the maturation period. Moreover, no significant uptake of the sugars was detected, since the total amount of fructose, glucose and sucrose remained constant. Our results indicate that the action of sucrose on embryo maturation is mostly achieved through an osmotic control.     

Cell wall invertase activity in cultured tobacco tissues

Changes in insoluble or cell wall invertase and soluble invertase activity were examined during callus induction from tobacco pith-phloem explants and during callus proliferation on sucrose, glucose and fructose as carbon sources, or on transfer from culture on the hexoses to sucrose. In all cases there was a growth independent transitory increase in cell wall invertase early in culture. The magnitude of the increase was greatest in the presence of sucrose. Cell wall invertase was found to possess catalytic activity in situ, whether or not the tissue was grown on sucrose. It is hypothesized that the transitory increase in cell wall invertase plays a role in sucrose hydrolysis during wound respiration, which takes place early in culture.     

Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving

Tissue culture media or aqueous sucrose solutions containing activated charcoal buffered to pH 5.5 and autoclaved did not undergo appreciable sucrose hydrolysis as reported. Rather, the extent of sucrose hydrolysis in media containing activated charcoal was found to be directly proportional to the hydrogen ion concentration (pH). This finding is consistent with the known mechanism of acid-catalyzed hydrolysis of acetals such as sucrose. Several types of charcoal were identified that acidified culture media to the extent that considerable acid-catalyzed sucrose hydrolysis occurred under autoclave conditions, making it appear as though activated charcoal was responsible for catalyzing sucrose hydrolysis. A simple mathematical expression was empirically derived that can be used to predict the extent of sucrose hydrolysis based on the post-autoclave pH of the media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

Actinidia deliciosa in vitro II. Growth and exogenous carbohydrates utilization by explants

Changes in sugar composition (sucrose, glucose and fructose) of medium, callus, stem and leaves of in vitro proliferating explants of Actinidia deliciosa C.F. Liang, Hayward were analyzed together with explant growth at 0, 15, 30, 45 and 60 days of culturing. Autoclaving hydrolyzes a small part of the initial sucrose of the medium into glucose and fructose. In presence of Actinidia explants the initial sucrose decreased to 32% after 15 days of culturing, to 4% after 30 days and to 0.08% at the end of the culture period (60 days). Sucrose increase in the explants did not parallel with its decrease in the medium. Sucrose presence in the explants was evident only during the last month of culturing. After 15 days of culturing a large increase of glucose and fructose was found in the medium but it did not equal the hydrolyzed sucrose. The level of these two monosaccharides remained stable in the medium until the 30th day, then significantly decreased in the second month of culture; neither were completely exhausted at the end of the culture. In the whole explant the highest amount of glucose and fructose was reached after 30 days of culturing.The balance of the three sugars in the medium-explant system, as % distribution of carbon atoms, showed a utilization throughout the whole culture period.Qualitative analyses performed on medium, callus and leaves at 0, 15, and 30 days of culturing revealed the presence of glucose and fructose only and no significant amounts of other hexoses or pentoses. Starch accumulation in the leaves was also observed throughout the culturing.Paper No. 724     

The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l^-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.     

Transport of glucose,fructose and sucrose by Streptanthus tortuosus suspension cells

Streptanthus tortuosus Kell. suspension cells will grow in a medium with sucrose as carbohydrate source. It was investigated whether the cells are able to take up sucrose or whether sucrose has to be hydrolyzed to glucose and fructose which eventually are taken up. The detailed quantitative analysis of sugar-uptake rates in the low concentration range up to 1 mM showed the following features: (i) There is definitely no sucrose-uptake system working in the low concentration range; any uptake of radioactivity from labelled sucrose proceeds via hydrolysis of sucrose by cell-wallbound invertase. (ii) Hexoses are taken up by two systems, a glucose-specific system with a K _m of 45 M and a high V _max for glucose and a K _m of 6 mM and a low V _max for fructose, and a fructosespecific system with a K _m of 500 M and high a V _max for fructose and a K _m of 650 M and a low V _max for glucose. (iii) There is a more than tenfold preference for uptake of the fructose derived from sucrose versus uptake of free fructose, with the result that the kinetic disadvantage of the fructoseuptake system compared to the glucose-uptake system is diminished if sucrose is supplied as the carbon source. It is speculated that invertase might work as an enzyme aiding in fructose transport.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - FW fresh weight     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. Grand Rapids

The timing of mobilisation of lipid, sucrose, raffinose and phytate in lettuce seeds (achenes) (cv. Grand Rapids) has been examined. These reserves (33%, 1.5%, 0.7%, 1.4% of achene dry weight, respectively) are stored mostly in the cotyledons. Except for a slight degradation of raffinose and increase in sucrose, there is no detectable reserve mobilisation during germination. The endosperm (8% of seed dry weight), which has thick, mannan-containing cell walls (carbohydrate, 3,4% of seed dry weight), is completely degraded within about 15h following germination. Mannanase activity increases about 100-fold during the same period and arises in all regions of the endosperm. Also during this period sucrose and raffinose are degraded and fructose and glucose accumulate in the embryo. The endosperm hydrolysis products are taken up by the embryo, and are probably used as an additional reserve to support early seedling growth. However, endosperm cell-wall carbohydrates, such as mannose, are not found as free sugars. Lipid and phytate are degraded in a later, second phase of mobilisation. Low levels of sucrose are present in the embryo, mostly in the cotyledons, and large amounts of fractose and glucose (14% of seedling dry weight at 3 days after sowing) accumulate in the hypocotyl and radicle. It is suggested that sucrose, produced in the cotyledons by gluco-neogenesis, is translocated to the axis and converted there to fructose and glucose.     

GROWTH AND DEVELOPMENT OF REPRODUCTIVE AND VEGETATIVE TISSUES OF BOUGAINVILLEA CULTURED IN VITRO AS A FUNCTION OF CARBOHYDRATE

Inflorescence meristems and vegetative tissues, excised from noninduced Bougainvillea ‘San Diego Red’ plants, were cultured in vitro in media containing either 3% fructose, glucose or sucrose as carbon sources. Growth and development of young leaves were equivalent whether sucrose or fructose was used whereas floret initiation on inflorescence meristems was much greater when fructose or glucose was the carbon sources. Brief (1-3 days) exposure of inflorescence meristems to fructose at the beginning of culture and subsequent transfer to sucrose did not increase development over continuous culture in sucrose. Longer exposures (4-7 days) to fructose with subsequent transfer to sucrose did, however, increase the percentage of meristems developing florets, but such treatment did not increase development to the same level as those exposed to fructose for the entire period in vitro. During the first 18 days of culture, growth of meristems in sucrose was linear while that in fructose was exponential. There was no difference in carbohydrate requirements for floret initiation on meristems excised from short-day induced or noninduced plants, suggesting that induction does not enhance the ability of meristems to utilize sucrose.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

The relationship between sucrose hydrolysis,sorbitol formation and mineral ion concentration during bioethanol formation using Zymomonas mobilis 2716

Summary Investigations into the relationship between sucrose hydrolysis, sorbitol formation and mineral ion concentration during bioethanol formation by Zymomonas mobilis 2716 revealed two distinct phenomena responsible for carbon flow diversion, a sucrose effect and a salt effect. Neither of the two phenomena affects sucrose hydrolysis, but they divert carbon flow of the fructose monomer leading to its own accumulation, sorbitol or oligosaccharide formation. Sucrose concentrations in excess of 15% (w/v) led to sorbitol formation, the level of which may exceed 2% (w/v) depending upon glucose accumulation during sucrose hydrolysis. Increasing mineral ion concentrations led initially to carbon losses and finally to fructose accumulation instead of sorbitol formation. This carbon loss can be corrected by the addition of invertase, which in turn leads to an increase in sorbitol, fructose and ethanol. Potassium and chloride are the dominant ions responsible for suppression of sorbitol formation and fructose uptake, encouraging oligosaccharide formation. These fructooligosaccharides must be of a type which can be converted to fructose, sorbitol and ethanol through the action of invertase. The requirement of invertase addition to prevent fructooligosaccharide formation is indirect evidence that Z. mobilis 2716 does not produce invertase.Offprint requests to: H. W. Doelle     

Screening <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (Spirulina) strains for heterotrophy

Thirty-five clonal, axenic Arthrospira strains were screened for their ability to grow heterotrophically on six carbon sources (20 mM). Glucose (34 strains) and fructose (24 strains) were the only substrates permitting growth in the dark. In some assays, however, not every replicate grew and, in at least one strain (D867), repeat assays over 2 years on material maintained in the light indicated that the ability to use fructose in the dark had become lost. Four further strains from other culture collections were compared, because they are presumed duplicates of three of the main set of strains; in at least three cases the duplicates of a pair differed in their ability to use fructose in the dark. In another comparison, where straight and helical morphotypes of the same strain could be compared, two of the four straight morphotypes (including one duplicate strain) grew with glucose in the dark, whereas none of the helical morphotypes did so. It is suggested that genetic drift has led to losses in the ability to grow heterotrophically in some strains.Ten of the main set of strains were tested for their ability to grow photoheterotrophically with four of the carbon sources (glucose, maltose, fructose, sucrose). All grew with glucose and maltose, but none with fructose or sucrose, in spite of the fact that eight (of this subset) grew with fructose in the dark. Sucrose led to most of a culture lysing, but often with short fragments of trichome surviving and subsequently giving rise to a normal culture. All the surviving cells in the transitory stage showed the presence of large intrathylakoidal granules, which had disappeared by the time that the culture had returned to a healthy state. Bleaching and recovery were more rapid at 70 than 10 μmol photon m⁻² s⁻¹. The presence of DCMU prevented this recovery. There was no bleaching when an inoculum of the recovered material was subcultured to medium with sucrose.