Application Of Sucrose Research Articles

Secondary Taste Neurons that Convey Sweet Taste and Starvation in the Drosophila Brain

The gustatory system provides vital sensory information to determine feeding and appetitive learning behaviors. Very little is known, however, about higher-order gustatory circuits in the highly tractable model for neurobiology, Drosophila melanogaster. Here we report second-order sweet gustatory projection neurons (sGPNs) in the Drosophila brain using a powerful behavioral screen. Silencing neuronal activity reduces appetitive behaviors, whereas inducible activation results in food acceptance via proboscis extensions. sGPNs show functional connectivity with Gr5a(+) sweet taste neurons and are activated upon sucrose application to the labellum. By tracing sGPN axons, we identify the antennal mechanosensory and motor center (AMMC) as an immediate higher-order processing center for sweet taste. Interestingly, starvation increases sucrose sensitivity of the sGPNs in the AMMC, suggesting that hunger modulates the responsiveness of the secondary sweet taste relay. Together, our results provide a foundation for studying gustatory processing and its modulation by the internal nutrient state.

AtCCR4a and AtCCR4b are Involved in Determining the Poly(A) Length of Granule-bound starch synthase 1 Transcript and Modulating Sucrose and Starch Metabolism in Arabidopsis thaliana.

Removing the poly(A) tail is the first and rate-limiting step of mRNA degradation and apparently an effective step not only for modulating mRNA stability but also for translation of many eukaryotic transcripts. Carbon catabolite repressor 4 (CCR4) has been identified as a major cytoplasmic deadenylase in Saccharomyces cerevisiae. The Arabidopsis thaliana homologs of the yeast CCR4, AtCCR4a and AtCCR4b, were identified by sequence-based analysis; however, their role and physiological significance in plants remain to be elucidated. In this study, we revealed that AtCCR4a and AtCCR4b are localized to cytoplasmic mRNA processing bodies, which are specific granules consisting of many enzymes involved in mRNA turnover. Double mutants of AtCCR4a and AtCCR4b exhibited tolerance to sucrose application but not to glucose. The levels of sucrose in the seedlings of the atccr4a/4b double mutants were reduced, whereas no difference was observed in glucose levels. Further, amylose levels were slightly but significantly increased in the atccr4a/4b double mutants. Consistent with this observation, we found that the transcript encoding granule-bound starch synthase 1 (GBSS1), which is responsible for amylose synthesis, is accumulated to a higher level in the atccr4a/4b double mutant plants than in the control plants. Moreover, we revealed that GBSS1 has a longer poly(A) tail in the double mutant than in the control plant, suggesting that AtCCR4a and AtCCR4b can influence the poly(A) length of transcripts related to starch metabolism. Our results collectively suggested that AtCCR4a and AtCCR4b are involved in sucrose and starch metabolism in A. thaliana.

A re‐assessment of sucrose signaling involved in cluster‐root formation and function in phosphate‐deficient white lupin (Lupinus albus)

Apart from substrate functions, a signaling role of sucrose in root growth regulation is well established. This raised the question whether sucrose signals might also be involved in formation of cluster-roots (CRs) under phosphate (Pi) limitation, mediating exudation of phosphorus (P)-mobilizing root exudates, e.g. in Lupinus albus and members of the Proteaceae. Earlier studies demonstrated that CR formation in L. albus was mimicked to some extent by external application of high sucrose concentrations (25 mM) in the presence of extremely high P supply (1-10 mM), usually suppressing CR formation. In this study, we re-addressed this question using an axenic hydroponic culture system with normal P supply (0.1 mM) and a range of sucrose applications (0.25-25 mM). The 2.5mM sucrose concentration was comparable with internal sucrose levels in the zone of CR initiation in first-order laterals of P-deficient plants (3.4 mM) and induced the same CR morphology. Similar to earlier studies, high sucrose concentrations (25 mM) resulted in root thickening and inhibition of root elongation, associated with a 10-fold increase of the internal sucrose level. The sucrose analog palatinose and a combination of glucose/fructose failed to stimulate CR formation under P-sufficient conditions, demonstrating a signal function of sucrose and excluding osmotic or carbon source effects. In contrast to earlier findings, sucrose was able to induce CR formation but had no effect on CR functioning with respect to citrate exudation, in vitro activity and expression of genes encoding phosphoenolpyruvate carboxylase, secretory acid phosphatase and MATE transporters, mediating P-mobilizing functions of CRs.

Application of abscisic acid (S-ABA) and sucrose to improve colour, anthocyanin content and antioxidant activity of cv. Crimson Seedless grape berries

Background and Aims In some years, the table grape cultivar Crimson Seedless does not reach the commercially acceptable level of red colour in Mediterranean climates, with negative consequences for the grower. Application of abscisic acid (S-ABA) and sucrose could be a tool for overcoming this problem. Methods and Results A commercial formulation of S-ABA (400 mg/L) and sucrose (10%) was applied to Crimson Seedless at the beginning of veraison and 5 days later in order to verify the effect on harvestable bunches, skin colour, chemical characteristics, anthocyanin content, antioxidant activity and S-ABA concentration either in the skin or the pulp of the berry. The application of S-ABA and sucrose improved the colour of the berries and increased the amount of harvestable bunches at the first pick (80–95%) compared with that of the control treatment (43%). Ripening attributes (total soluble solids, pH, titratable acidity) were not affected by the application of either S-ABA or sucrose. The S-ABA concentration in skin and pulp increased significantly after the treatments and at first harvest. Abscisic acid increased the concentration of anthocyanins and showed a positive effect on the antioxidant activity. Conclusion Abscisic acid and sucrose improved the colour of the berry skin, with improved results obtained when S-ABA was applied alone and at the beginning of veraison. Sucrose, although to a less extent, was also effective in improving skin colour and increasing the number of harvestable bunches at the first pick. Significance of the Study This study provides new information about the positive effect of either S-ABA or sucrose on colour, anthocyanin concentration and antioxidant activity of the berry. Moreover, bunches could be harvested in fewer picks with important economic consequences for the grower and with an S-ABA concentration in the berry within the physiological values.

Nebulization of Active Pharmaceutical Ingredients with the eFlow®rapid: Impact of Formulation Variables on Aerodynamic Characteristics

Nebulization of active pharmaceutical ingredient (API) solutions is a well-established means to achieve pulmonary drug deposition. The current study identified the impact of formulation variables on the aerosolization performance of the eFlow(®) rapid with special respect to optimized lung application. API formulations (including excipient-supplemented samples) were investigated for physicochemical properties, then nebulized using vibrating-mesh technology. The generated aerosol clouds were analyzed by laser diffraction. Aerosol deposition characteristics in the human respiratory tract were estimated using an algebraic model. Remarkable effects on aerosolization performance [i.e., mass median aerodynamic diameter (MMAD)] of API solutions were obtained when the sample conductivity (by API concentration and type, sodium chloride addition) and dynamic viscosity (by application of sucrose and poly(ethylene glycol) 200) were elevated. A similar influence was observed for a decline in surface tension (by ethanol addition). Thus, a defined adjustment of formulation parameters allowed for a decrease of the MMAD from ∼ 8.0 μm to values as small as ∼ 3.5 μm. Consequently, the pattern and efficiency of aerosol deposition in the human respiratory tract were improved. In conclusion, identification of physicochemical variables and their way of influencing vibrating-mesh nebulization has been provided to deliver a platform for tailoring aerosol characteristics and thus, advancing pulmonary therapy.

Sugars take a central position in plant growth, development and, stress responses. A focus on apical dominance.

Sugars take a central position in plant growth, development and, stress responses. A focus on apical dominance.

Effects of nano-silver and sucrose applications on cut flower longevity and quality of tuberose (Polianthus tuberosa).

Tuberose (Polianthus tuberosa) is a commercially important cut flower; but its longevity and quality characteristics are compromised by stress during storage and transportation. Thus, it is important to determine the most appropriate preservative solution to best maintained cut flowers. Tests were done to examine the effects on of nano-silver (NS) (0, 15, 30 and 45 mg L-1) and sucrose (0, 4 and 8%) on the quality properties of fresh weight, relative water content, water uptake, total soluble solids, wilting percentage, flower opening and flower longevity. Results showed that averages of evaluations for flower longevity, open flower and stem diameter, fresh weight and relative water content increased under sucrose application and that those for while flower longevity, open flower and stem diameter, water uptake, fresh weight and relative water uptake were increased by NS application. However, applications of 4% sucrose or 45 mg L-1 NS treatments enhanced all measured parameters, with only a few exceptions. These parameters were further increased under the treatment of sucrose at 4% combined with 45 mg L-1 NS, suggesting that the quality of tuberose cut flowers can be improved by treatment with a combination of sucrose and NS.

Salty taste deficits in CALHM1 knockout mice.

Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt.

Low sink-induced stomatal closure alters photosynthetic rates of source leaves in beans as dependent on H2O2 and ABA accumulation in guard cells

Low sink demand provided by pod removal and stem girdling of beans (Vicia faba, cv. Daqingshan) (-Sink) induced a significantly lower net photosynthetic rate (Pn), stomatal conductance (gs), internal CO2 concentration (Ci), and transpiration rate (E) compared with pod and root sink retention (CK). This depression in Pn was due to stomatal limitation. Low sink demand of -Sink plants resulted in a higher leaf sucrose content, but a lower sucrose content in guard cells. Moreover, the significant accumulation of H2O2 and ABA were observed in both leaves and guard cells of -Sink plants. The most intensive electron dense deposit of cerium perhydroxides, produced by H2O2 reaction with cerium chloride, was present in the cell walls, especially the dorsal walls of guard cells. Immunogold electron-microscopy localization of ABA showed that ABA was distributed in ventral walls of guard cells and the intercellular space of mesophyll cells of -Sink leaves in contrast to CK plants. Application of exogenous sucrose to isolated bean leaves increased H2O2 and ABA contents. H2O2 and ABA in leaves was likely generated by two independently regulated pathways, each affected by the high sucrose concentration induced by low sink demand. Increased sucrose in leaves in response to low sink demand may have caused the increase of H2O2 and ABA, and their accumulation in mesophyll cells and guard cells was likely the primary cause for stomatal closure under low sink demand.

Analysis of the Sensory and Hedonic Impacts of Sweet and Bitter Tastes in Perinatally Underfed Rats

Newborn rats discriminate tastes and generate gustofacial response (GFR) because the neuronal substrate is already operating. The oral application of sucrose or quinine hydrochloride produces a specific GFR. We analyzed the effects of perinatal undernutrition on the GFR development of rats at two cue concentrations. In the undernourished group, pregnant dams received different percentages of a balance diet. After birth, prenatally underfed pups continue the undernourishment by remaining for 12 h with a foster dam, and for 12 h with a nipple-ligated mother. Cues were presented as a single droplet of sucrose, sodium chloride, or quinine at low or high concentrations onto the lips at postnatal days (PDs) 1 and 3, and mouth-opening (MOF) and lip-licking frequencies (LLF) were noted. On PD 1 the undernourished group showed smaller MOF increases in response to low salt and quinine stimuli than the controls but no differences at high concentrations. On PD 3, both low and high concentrations of the sucrose and quinine cues significantly increased the MOF in the underfed compared to the control group. Low but not high salt decreased LLF on PD1 in the underfed compared to the control group. On PD 3 the undernourished pups showed significant increases of LLF with low quinine compared with the control rats, but the reverse was observed with high quinine. These data suggest that perinatal undernutrition affects the development of the sensory and hedonic aspects of taste causing changes in GFR expression.

Newly Identified Characteristic of Taste Avoidance Conditioning with High Voltage as an Unconditional Stimulus in <i>Lymnaea Stagnalis</i>

Based on the newly established taste avoidance conditioning paradigm, Lymnaea can learn the temporal sequence of sucrose application and subsequent applied high voltage with small current resulting in significant suppression of feeding response to sucrose. The most efficient condition to acquire the long-term memory (LTM) is obtained by 15 paired presentations of a conditional stimulus (CS) and an unconditional stimulus (US). This repetition number for LTM acquisition is different among the taste avoidance conditioning employed as US, such as KCl, mechanical tapping and electrical high voltage, however the behavioral modification is thought to activate the same neuronal circuit via RPeD11 which is the inter neuron to control the whole-body withdrawal response. All of these USs are thought to be recognized as a noxious stimulus sensing at RPeD11.

Sucrose induces expression of the sorbitol‐6‐phosphate dehydrogenase gene in source leaves of loquat

Rosaceae fruit trees use sorbitol and sucrose as translocating sugars and the sorbitol-to-sucrose ratio in source leaves determines apple fruit quality. Here, we investigate the effects of sugars on the expression of genes encoding key photosynthetic enzymes, including sorbitol-6-phosphate dehydrogenase (S6PDH, EC 1.1.1.200), sucrose phosphate synthase (SPS, EC 2.4.1.14), and ADP-glucose pyrophosphorylase (ADPGPPase, EC 2.7.7.27) to understand the sugar-signaling mechanism in Rosaceae fruit trees. Mature leaf-petiole cuttings of loquat (Eriobotrya japonica Lindl. cv. Mogi) were supplied with a water, sorbitol or sucrose solution for 2 days at 20°C. The relative levels of the transcripts were analyzed by real-time polymerase chain reaction (PCR). S6PDH transcription was decreased by sorbitol but drastically increased by sucrose. SPS and ADPGPPase large subunit transcription were decreased by sucrose and sorbitol. The simultaneous application of sorbitol and sucrose revealed that S6PDH transcription increased in a dose-dependent manner with sucrose. These results show that both sorbitol and sucrose work as signaling molecules in source organs of Rosaceae fruit trees. These trees have mechanisms to positively keep sorbitol as the dominant translocating sugar, suggesting that sorbitol plays an important role in their survival strategy. Effects of various sugars on S6PDH expression were investigated. Palatinose, a sucrose analog, increased S6PDH transcription much more drastically than sucrose. Mannose and 3-O-methylglucose, glucose analogs, also increased S6PDH transcription; however, glucose did not. Models of sugar signaling in source organs of Rosaceae fruit trees are discussed.

High voltage with little current as an unconditional stimulus for taste avoidance conditioning in Lymnaea stagnalis

A new and better taste avoidance conditioning paradigm for Lymnaea has been developed that replaces the previously used tactile unconditional stimulus (US) with an brief electrical stimulus (1000V, 80μA), while continuing to use a sucrose application to the lips as the conditional stimulus (CS). With 15 paired CS–US presentations on a single day, we were able to elicit both short-term memory (STM) and long-term memory (LTM). The LTM persisted for at least one week. While STM was elicited with 5, 8, or 10 paired presentations of the CS–US on a single day, LTM was not. The new US used here was more consistent than the previously used US, and this stimulus consistency may explain why 15 paired CS–US presentations now result in LTM formation.

Expression of the 1-SST and 1-FFT genes and consequent fructan accumulation in Agave tequilana and A. inaequidens is differentially induced by diverse (a)biotic-stress related elicitors

The expression of genes coding for sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100), both fructan biosynthesizing enzymes, characterization by TLC and HPAEC-PAD, as well as the quantification of the fructo-oligosaccharides (FOS) accumulating in response to the exogenous application of sucrose, kinetin (cytokinin) or other plant hormones associated with (a)biotic stress responses were determined in two Agave species grown in vitro, domesticated Agave tequilana var. azul and wild A. inaequidens. It was found that elicitors such as salicylic acid (SA), and jasmonic acid methyl ester (MeJA) had the strongest effect on fructo-oligosaccharide (FOS) accumulation. The exogenous application of 1mM SA induced a 36-fold accumulation of FOS of various degrees of polymerization (DP) in stems of A. tequilana. Other treatments, such as 50mM abscisic acid (ABA), 8% Sucrose (Suc), and 1.0mgL−1 kinetin (KIN) also led to a significant accumulation of low and high DP FOS in this species. Conversely, treatment with 200μM MeJA, which was toxic to A. tequilana, induced an 85-fold accumulation of FOS in the stems of A. inaequidens. Significant FOS accumulation in this species also occurred in response to treatments with 1mM SA, 8% Suc, and 10% polyethylene glycol (PEG). Maximum yields of 13.6 and 8.9mg FOS per g FW were obtained in stems of A. tequilana and A. inaequidens, respectively. FOS accumulation in the above treatments was tightly associated with increased expression levels of either the 1-FFT or the 1-SST gene in tissues of both Agave species.

Integrated transcriptional and phytochemical analyses of the flavonoid biosynthesis pathway in Epimedium

Epimedium is a widely used traditional Chinese medicinal plant due to its high content of health-promoting components such as epimedin A, epimedin B, epimedin C, and icariin (hereafter designated as bioactive compounds); however, the biosynthesis of these bioactive compounds in Epimedium remains to be elucidated. In this study, E. sagitattum flavonoid genes, including three copies of chalcone synthase, two copies of dihydroflavonol 4-reductase, and a single copy of each of chalcone isomerase, flavanone 3 hydroxylase, flavonol synthase, and anthocyanidin synthase, have been isolated. In addition, several transcription factors involved in flavonol, anthocyanin, and proanthocyanin pathways, were identified from an Epimedium 454 EST database. Gene expression profiles and the levels of bioactive compounds in light-stressed E. pubescens leaves in response to phytohormones [abscisic acid (ABA), gibberellic acid (GA3), indole-3-acetic acid (IAA), salicylic acid (SA)] and sucrose were investigated. All bioactive compounds were significantly elevated by phytohormone and sucrose application except for epimedin B in SA-treated samples and epimedin C following GA3 and sucrose treatment. Our results suggest that phytohormone and sucrose treatments offer a method for promoting the accumulation of bioactive compounds in Epimedium.

8. Transition into and out of a seizure in the hippocampal CA3 region: A failure of presynaptic release

How the brain transitions into and out of a seizure is mysterious. Using the intact mouse hippocampus preparation, recurrent seizure-like events (SLEs) in low Mg 2+ /high K + perfusate were measured in the CA3 region. The SLE was characterized by a “preictal phase”, which abruptly turns into a higher frequency “ictal” phase. Blockade of GABA A receptors shortened the preictal phase, abolished interictal bursts, attenuated the slow preictal depolarization, but with no effect on the ictal duration. On the other hand, SLEs were blocked by glutamate receptor blockade. In CA3 pyramidal cells and stratum oriens non-fast and fast spiking interneurons, recurrent GABAergic inhibitory postsynaptic currents (IPSCs) predominated interictally and during the early preictal phase, synchronous with extracellularly measured recurrent field potentials (FPs). These IPSCs then decreased to zero or reversed polarity by the onset of the higher frequency ictus. However postsynaptic muscimol-evoked GABA A responses remained intact. Simultaneously, excitatory postsynaptic currents (EPSCs) synchronous with the FPs, markedly increased to a maximum at the ictal onset. The reversal potential of the compound postsynaptic currents (combined simultaneous EPSCs and IPSCs: PSCs) became markedly depolarized during the preictal phase, whereas the muscimol-evoked GABA A reversal potential remained unchanged. During the late preictal phase, interneuronal excitability was high, but IPSCs, evoked by local stimulation, or osmotically by hypertonic sucrose application, were diminished, disappearing at the ictal onset. EPSCs evoked by hypertonic sucrose application, were maximal at ictal onset, disappearing at the end of the ictus. We conclude that the interictal and early preictal states are dominated by GABAergic activity, with the onset of the ictus heralded by exhaustion of presynaptic release of GABA, and unopposed increased glutamatergic responses. The ictus stops when presynaptic release of glutamate is exhausted. Supported by CIHR.

Decreased carbon limitation of litter respiration in a mortality-affected piñon–juniper woodland

Abstract. Microbial respiration depends on microclimatic variables and carbon (C) substrate availability, all of which are altered when ecosystems experience major disturbance. Widespread tree mortality, currently affecting piñon–juniper ecosystems in southwestern North America, may affect C substrate availability in several ways, for example, via litterfall pulses and loss of root exudation. To determine piñon mortality effects on C and water limitation of microbial respiration, we applied field amendments (sucrose and water) to two piñon–juniper sites in central New Mexico, USA: one with a recent (< 1 yr), experimentally induced mortality event and a nearby site with live canopy. We monitored the respiration response to water and sucrose applications to the litter surface and to the underlying mineral soil surface, testing the following hypotheses: (1) soil respiration in a piñon–juniper woodland is water- and labile C-limited in both the litter layer and mineral soil; (2) piñon mortality reduces the C limitation of litter respiration; and (3) piñon mortality enhances the C limitation of mineral soil respiration. Litter respiration at both sites responded to increased water availability, yet surprisingly, mineral soil respiration was not limited by water. Consistent with hypothesis 2, C limitation of litter respiration was lower at the recent mortality site compared to the intact canopy site. Applications to the mineral soil showed evidence of reduction in CO2 flux on the girdled site and a non-significant increase on the control. We speculate that the reduction may have been driven by water-induced carbonate dissolution, which serves as a sink for CO2 and would reduce the net flux. Widespread piñon mortality may decrease labile C limitation of litter respiration, at least during the first growing season following mortality.

Exogenous Sugars Involvement in Senescence and Ethylene Production of Tree Peony 'Luoyang Hong' Cut Flowers

Sugars play important roles in petal senescence of cut flowers. In the Expt. 1 of this study, the effects of different concentrations of glucose (60, 90, and <TEX>$120g{\cdot}L^{-1}$</TEX>) and sucrose (30, 60, and <TEX>$90g{\cdot}L^{-1}$</TEX>) application on the vase life, rate of flower diameter increase, rate of flower weight increase and ethylene production of cut tree peony (Paeonia suffruticosa 'Luoyang Hong') were evaluated. At the earlier stage, treatments of different concentrations of glucose and sucrose all retarded the process of flower opening and inhibited the increase of flower diameter and weight, while senescence of flowers fed with different concentrations of glucose was delayed at later stage. Flowers treated with <TEX>$90g{\cdot}L^{-1}$</TEX> glucose displayed the longest vase life, which showed significant difference (P < 0.05) from those of flowers with the control and sucrose treatments. All treatments with glucose or sucrose not only retarded the decrease of flower diameter and weight, but also suppressed the ethylene production at the earlier stage and delayed the peak of ethylene evolution. In order to study the effect of exogenous sugar on the postharvest response of cut tree peony to ethylene, Expt. 2 was conducted. Cut flowers were treated with <TEX>$90g{\cdot}L^{-1}$</TEX> glucose for 4 hours before (GE) or after (EG) exposed to <TEX>$10{\mu}L{\cdot}L^{-1}$</TEX> ethylene for 4 hours. Generally, the opening process of flowers with GE and EG treatments was similar to that of the control, however GE treatment delayed flower senescence. Both GE and EG treatments improved flower diameter and weight, and GE treatment delayed the time of flower weight decrease. Besides, GE delayed climacteric ethylene evolution for 8 hours. All above suggest that exogenous sugars delay tree peony 'Luoyang Hong' cut flower senescence and extend flower vase life through their roles in the decrease of water loss and the suppression of sensitivity to ethylene and ethylene production.

Study on Cryo-preservation of Theileria annulata Schizont Infected Cell Line Vaccine Strain S15

The p urpose of this study was to examine the potential application of Sorbitol, Sucrose and Gelatin in combination with glycerol in order to Theileria annulata infected cell line cryo-preservation. The efficiency of the freezing methods was compared by assessing the viability and plating efficiency of cells after resuscitation, dilution and equilibration in cell culture medium. As a result, the conventional cryopreservative medium containing 5% PEG treated Bovine Serum and 10% glycerol successfully cryopreserved cell as efficiently as the standard medium containing 80% FBS and 10% glycerol and was superior to all thirteen different experimental cryo-preservative media. A crucial result was obtained among the thirteen different serum-free media which showed that the 0.2M sorbitol+10% glycerol method yielded cells with viability of 54% and good plating efficiency results. It was concluded that the conventional method would be the best and ideal for the cryo-preservation and storage of Theileria annulata-infected cells for vaccine purposes to date.

Effects of sugars on the growth and chlorophyll content in excised tulip stem in the presence of Indole-3-acetic acid

ABSTRACT The purpose of this study was to clarify the effect of sucrose on auxin-induced growth of stem excised from growing tulips and excised directly from cooled and not cooled bulbs, and on the growth of excised IV internode from growing plants in the presence of auxin. In all cases flower bud was replaced by IAA (indole-3- acetic acid, 0.1%, w/w in lanolin) and basal part of excised segments of stem was kept in distilled water or in solution of various sugars at different concentrations. IAA-induced growth of excised stems isolated from growing tulips was inhibited by sucrose at concentrations of 5.0% and 10.0%, but sucrose at 1.25% and 2.5% did not. Sucrose at all concentrations used evidently delayed senescence and increased chlorophyll contents in excised stems in the presence of IAA. Sucrose induced stiffing in isolated stems in the presence of IAA, and much less infective by pathogen in comparison to stem treated with IAA only. Mannitol and sorbitol at concentrations of 5.0% and 10.0% substantially inhibited IAA-induced growth of stem segments. Stem segments excised from cooled and not cooled tulip bulbs were more sensitive than those isolated from growing shoots due to application of sucrose and glucose; more inhibitory effect was observed. Sucrose at concentrations of 5.0% and 10.0% only slightly inhibited growth of IV internode treated with IAA and all concentrations of sucrose (1.25%, 2.5%, 5.0% and 10.0%) substantially increased chlorophyll content. The possible mode of actions of sucrose interacting with auxin to regulate stem growth is also discussed although sugar response is complicated by the fact that plants have multiple sugar-response pathways.