OBJECTIVETo research the optimal conditions for the callus induction of anther culture and the plant regeneration of Angelica dahurica var. formosana.

METHODCallus was induced from the anther of A. dahurica from Sichuan province on a MS medium. The effects of callus induction and plant regeneration of different pretreatment hours under low temperature (4 degrees C), different culturing conditions under darkness and illumination, and different culture with different hormone contents and ratios were studied.

RESULTThe results showed that A. dahurica anthers without low temperature pretreatment reached the highest induction rate then under the pretreatment under low temperature (4 degrees C) for two days. The optimal culturing condition was under the darkness. The culturing efficiency reached 38.89% on the medium of MS + 2.0 mg x L(-1) 2,4-D + 1.0 mg x L(-1) 6-BA. The optimum medium for differentiate anther callus was MS + 0.5 mg x L(-1) NAA + 1.5 mg x L(-1) KT + 10 mg x L(-1) AgNO3. 1/2MS medium supplemented with 0.5 mg x L(-1) IBA could well promote seedings to take roots.

CONCLUSIONAn efficient system for callus induction of anther culture and plant regeneration of A. dahurica was preliminarily established.

Angelica ; drug effects ; growth & development ; Culture Media ; chemistry ; pharmacology ; Flowers ; Tissue Culture Techniques ; methods

To demonstrate the fructose metabolism pathway in Bifidobacterium Longum NCC2705 and to construct its fermentation model, we explored the comparative proteome cultivating the strain on glucose or fructose, based on a proteomic reference map of B. longum NCC2705 constructed earlier. Then, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and electro-spray ionization tandem mass spectrometry (ESI-MS/MS) for differently expressed proteins identification. Furthermore, with semi-quantitative RT-PCR we determined the distinctively expressed proteins at the level of transcription. Proteomic comparison of glucose- and fructose-grown cells demonstrated much similarity. On the page of fructose there were all the enzymes and proteins that exist during the process of glucose degradation. We observed a greater variation of more than three-fold for the identified 9 spots representing 5 protein entries by MALDI-TOF MS. The sugar-binding protein specific to fructose (BL0033) and an ABC transporter ATP binding protein (BL0034) showed higher expression level from cells grown on fructose. It was also determined by semi-quantitative RT-PCR subsequently. BL0033 time course and concentration experiments showed that the induction time correlated to higher fructose concentration, and increased expression of BL0033. Fructose was catabolized via the same degradation pathway as glucose at the level of proteomics. BL0033 was induced by fructose. All results suggest that the uptake of fructose into the cell may be conducted by a specific ABC transport system, in which BL0033 and BL0034 as components might have played an important role.
Bifidobacterium ; chemistry ; genetics ; metabolism ; Culture Media ; Fermentation ; Fructose ; pharmacology ; Glucose ; pharmacology ; Proteome ; analysis ; genetics ; Proteomics ; methods

We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.
Calcimycin ; pharmacology ; Calcium ; chemistry ; Calcium Channel Blockers ; pharmacology ; Calcium Ionophores ; pharmacology ; Cell Culture Techniques ; Culture Media ; chemistry ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; metabolism ; Verapamil ; pharmacology

OBJECTIVETo investigate the effects of physical and chemical factors on callus growth and phillyrin contents of F. suspensa.

METHODThe cell growth index and phyllirin yield in different culture condition such as different plant hormones mixed, mediums, light and dark were compared. HPLC was used to examine phillyrin contents.

RESULT AND CONCLUSIONGrowth cycle of cells is twenty-eight days. During the course of callus growth, the processes of phillyrin biosynthesis were parallel with the cell growth. The optimum medium is MS. The optimum hormones concentrations are 1 mg.L-1 2,4-D, 0.5 mg.L-1 6-BA and 0.5 mg.L-1KT. The cell culture in light is more suitable than that in dark.

Culture Media ; Culture Techniques ; Forsythia ; chemistry ; cytology ; metabolism ; Glucosides ; biosynthesis ; Lighting ; Plant Growth Regulators ; pharmacology ; Plants, Medicinal ; chemistry ; cytology ; metabolism

OBJECTIVETo establish a protocol of rapid clonal propagation of Saussurea involucrate by using embryo as explants.

METHODMS medium was used as basal medium and BA and NAA were supplemented to find out the optimal hormone combinations for adventitious buds initiation, adventitious bud multiplication and rooting.

RESULTAll embryo explants started to grow adventitious buds within 45 days when they were cultured on the media supplemented with 0.02-0.1 mg x L(-1) NAA and 2 mg x L(-1) BA; The adventitious buds multiplicated within 30 days when they were transferred to the media containing 0.1-0.2 mg x L(-1) NAA and 2-3 mg x L(-1) BA; 92% of the adventitious buds rooted well after they were planted on the MS medium containing macroelements at half strength and 0.4 mg x L(-1) NAA for 25 days. The regenerated plantlets grew well after they were transplanted and the survival rate was up to 70%.

CONCLUSIONPlantlets of S. invducrate regenerated high frequence through adventitious bud.

Culture Media ; Plant Growth Regulators ; pharmacology ; Plants, Medicinal ; chemistry ; Regeneration ; Saussurea ; growth & development ; Tissue Culture Techniques ; methods

We studied the effects of several medicinal insects on biosynthesis of polysaccharides from Ganoderma lucidum in submerged culture. The results showed that the medicinal insect, Catharsius molossus at 5 g/L significantly promoted the biosynthesis of intracellular polysaccharides (IPS) and extracellular polysaccharides (EPS) of G. lucidum, and compared with control, IPS and EPS yields markedly enhanced from (1.93 +/- 0.09) g/L to (2.41 +/- 0.12) g/L and (520.3 +/- 20.2) mg/L to (608.9 +/- 20.2) mg/L, respectively (P < 0.05). Both IPS and EPS consisted of five kinds of components, and IPS-1 and EPS-1 were the major components of IPS and EPS, respectively. Further separation studies showed that IPS-1 was made up of three single compounds, while EPS-1 was made up of two single compounds. There were no new components in both IPS and EPS obtained from G lucidum in submerged culture by the addition of the insect, C. molossus, suggesting the biosynthetic pathways of the major components of IPS and EPS had not been changed.
Animals ; Cockroaches ; chemistry ; Culture Media ; chemistry ; Fermentation ; Materia Medica ; pharmacology ; Polysaccharides ; biosynthesis ; chemistry ; Reishi ; growth & development ; metabolism

Sterols are one of the active classes of compounds in Inonotus obliquus for their effective therapy of many diseases. In field environment, this fungus accumulates large amount of sterols. In cultured mycelia, however, this class of compounds is less accumulated. For analyzing the factors responsible for differing sterol composition, the field-grown and cultured mycelia were extracted with 80% ethanol at room temperature and total sterols were prepared using silicon gel column chromatography followed by identification using either GC-MS or spectroscopic methods. For culturing Inonotus obliquus, the seed culture was grown either in basic medium consisting of glucose (2%), yeast extract (0.5%), KH2PO4 (0.01%), MgSO4.7H20 (0.05%) and distilled water at pH 6.5, or the basic medium supplemented with serial concentrations of AgNO3. The results indicated that field-grown mycelia contained lanosterol and inotodiol (comprised 45. 47% and 25. 36% of the total sterols, respectively) and other 10 sterols (comprising the remaining 30.17%) including ergosterol biosynthetic intermediates such as 24-methylene dihydrolanosterol, 4,4-dimethylfecosterol, 4-methyl fecosterol, fecosterol and episterol. Column chromatography also led to the isolation of lanosterol, Inotodiol, trametenolic acid, foscoparianol B and a new triterpenoid foscoparianol D in field-grown mycelia. In comparison, the cultured mycelia only contained three sterols with ergosterol as the predominant one (82.20%). Lanosterol only accounted for 3.68%. Supplementing Ag+ into the culture at 0.28 micromol x L(-1) greatly enhanced content of lanosterol (accounting for 56.81%) and decreased the content of ergosterol (18.5%) together with the presence of intermediates for ergosterol biosynthesis. These results suggested that the sterol composition in mycelia of the fungus can be diversified by supplementing substances inhibiting enzymatic process towards the synthesis of ergosterol. Harsh growth conditions in field environment (i.e. temperature variation, UV irradiation etc.) can delay the synthesis of ergosterol and hereby diversify the sterol composition in the mycelia of Inonotus obliquus.
Basidiomycota ; chemistry ; growth & development ; Culture Media ; pharmacology ; Culture Techniques ; Ergosterol ; biosynthesis ; Gas Chromatography-Mass Spectrometry ; Lanosterol ; analogs & derivatives ; biosynthesis ; Mycelium ; chemistry ; Silver Nitrate ; pharmacology

OBJECTIVETo select optimal media and conditions for the tissue culture of Dendrobium lituiforum.

METHODThe basic media, light illumination condition, phytohormone, and natural aqueous extracts. were tested and compared. The chlorophyll contents and soluble protein contents were measured.

RESULTN6 medium was suitable for embryo germination and growth. 1/2 MS + NAA 0.5 mg x L(-1) cordd be used as the optimal protocorm multiplying media. Culture 20 days in darkness in advance, and then in light is useful to embryo germinate. 1/2 MS+ NAA 0-0.5 mg x L(-1) is beneficial to protocorm multiplication largely. N6 + 6-BA 2.0 mg x L(-1) + NAA 0.5 mg x L(-1) + 10% potato juice is useful to protocorm differentiation. Phytohormone and potato juice added to the media promoted chlorophyll content and souble protein content. N6 + NAA 0.5 mg x L(-1) + 10% banana juice is the best medium for plantlet rootage and strengthening.

CONCLUSIONConcentration of inorganic salt, nitrogen source and illumination condition are important to embryo germination and growth. The nitrogen source type has effect on the protocorm multiplication. The chlorophyll contents and souble protein contents may be index to indicate the growth condition of plantlets, which can help to select the optimal media.

Chlorophyll ; analysis ; Culture Media ; Dendrobium ; chemistry ; growth & development ; Light ; Plant Growth Regulators ; pharmacology ; Plant Proteins ; analysis ; Plants, Medicinal ; chemistry ; growth & development ; Tissue Culture Techniques

OBJECTIVETo study cortical neuron injury following recurrent epileptiform discharges induced by magnesium-free treatment in vitro.

METHODSCultured embryo cortical neurons were exposed to magnesium-free media for 3 h, then they were returned to regular media containing normal level magnesium. At different time after Mg(2+)-free treatment, trypan blue staining and determination of LDH activity were used to determine the cell viability, flow cytometry was applied to measure neuronal apoptosis, and MTT assay to study metabolic rate.

RESULTS(1) Neuronal morphology on light microscopy following Mg(2+)-free treatment showed that there were no prominent alterations. (2) At different time (6, 12, 72 h) after Mg(2+)-free treatment, neuronal viability by trypan blue staining and LDH activity showed modest changes compared with time-matched control in different culture days (6, 12, 17 d) (P > 0.05). (3) Cell apoptosis increased mildly at different time after Mg(2+)-free treatment in neurons cultured for different days, but the increase was not significant (P > 0.05). (4) Metabolic rate decreased at 6 h after Mg(2+)-free treatment (P < 0.05) in neurons cultured for 6 d, and was 86.4% of that of the control; while the rate at 24 h in neurons cultured for 12 d and 17 d also decreased (P < 0.05), being 78.7% and 70.9%, respectively, of that of the control.

CONCLUSIONSThese findings demonstrated that the injury occurred on cultured cortical neurons caused by magnesium-free-treatment-induced recurrent epileptiform discharges was mainly functional and relatively mature neurons displayed more severe and much later mitochondrial function impairment than immature neurons.

Animals ; Cerebral Cortex ; embryology ; Culture Media ; chemistry ; pharmacology ; Culture Techniques ; Magnesium ; pharmacology ; Neurons ; drug effects ; pathology ; Rats ; Rats, Wistar ; Seizures ; physiopathology

Chitinases were produced by a lot of microorganisms. Chitinase gene expression in most of the chitinase producing bacteria was inducible by chitin. Low levels of chitinase were observed in the presence of glucose. To date, however, the regulation of such chitinase gene in Bacillus thuringiensis had not been well studied. In this paper, all 77 Bacillus thuringiensis strains were grown in the medium with or without chitin. We measured quantitatively the chitinase activity of the cultures. Moreover, we investigated the suppressive effect of glucose on chitinase of 4 strains. Also we studied the relationship between chitin induction and glucose suppression on chitinase. This investigation demonstrated that all tested B. thuringiensis strains could produce chitinase without chitin. After induction, the chitinolytic activity of 31 tested strains had no obvious response to the inducer, whereas 44 stains increased in different degree. Among these strains, most of them did not markedly increase the levels of chitinase, and many stains simultaneously displayed the expression mode of inducible and constitutive. The glucose inhibited the inductive effect of chitin, but it could not inhibit the basal expression of chitinase. Two strains No. 38 and No. 75 belonged to different expression types. But we just found several different bases in the regulatory region of chitinase genes chiA and chiB from them.
Bacillus thuringiensis ; enzymology ; growth & development ; Base Sequence ; Chitin ; pharmacology ; Chitinases ; biosynthesis ; genetics ; Culture Media ; chemistry ; Culture Techniques ; Glucose ; pharmacology ; Molecular Sequence Data ; Polymorphism, Genetic