Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

Effect of Added Iron on the Formation of Clostridial Inhibitors

Inhibition of Clostridium botulinum by nitrite was potentiated by the addition of Fe(II) or Fe(III) to the culture medium. The effect of iron was more pronounced when nitrite was added after autoclaving.     

Micropropagation of gerbera using chlorine dioxide (ClO2) to sterilize the culture medium

One of the alternatives to autoclaving culture media is chemical sterilization, which may cause fewer changes to the chemical composition of the media. In this study, the effect of chemical sterilization by inclusion of chlorine dioxide (ClO₂) in the culture medium on the in vitro development of gerbera (Gerbera jamesonii) cv. AL101, cultured at different stages of micropropagation, was evaluated. The following five concentrations of ClO₂ were tested: 0%, 0.0025%, 0.0050%, 0.0075%, and 0.010%. Autoclaved medium was used as the control. ClO₂ in the culture medium reduced contamination at rates comparable to autoclaving when tested at three stages of the culture process: in vitro establishment, multiplication, and rooting. Plantlets grown in culture media sterilized with ClO₂ showed similar or better development than those grown in autoclaved culture medium. Use of 0.0025% ClO₂ to sterilize the culture medium resulted in better plantlet development than autoclaved medium, regardless of the stage of micropropagation.     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

Enhanced somatic embryogenesis and plant regeneration in leaf explant cultures of Ostericum koreanum on medium of varying pH

The leaf explants of Ostericum koreanum were cultured on MS medium supplemented with 5.37 M NAA and 0.44 M BA and did not need transfer to growth regulator–free medium for somatic embryogenesis. The pH level of medium dropped after autoclaving and at the beginning of explant culture, then rose back to the normal pH level of medium. The low pH level of medium, pH 4.0 or 4.3, before autoclaving rose to pH 5.2 or 5.3 and pH 6.1 or 6.2 after the 1 and 8 weeks from culture initiation, respectively, and this level was variable around pH 5–pH 6 during culture period. The explants were exposed to low pH for only several days at the early period of culture. On medium of pH 4.3, the production of somatic embryos was enhanced to six times in comparison with that on medium of pH 5.8. The average regeneration rate of total somatic embryos produced on medium of low pH was over 10% higher than that at pH 5.8. The regeneration of cup-shaped embryos was improved from 33% on medium of pH 5.8 to 67% on medium of pH 4.3. Therefore, the production and regeneration of somatic embryos were enhanced by the temporary exposure of leaf explant to medium of low pH, even though somatic embryogenesis substantially occurred on medium of nearly routine pH.     

Biogenesis of methane in primate dental plaque

Dental plaque samples collected from monkeys (Macaca mulatta) were found to contain a large amount of dissolved methane gas (0.6 nmol CH4/mg wet wt plaque). Enrichment cultures inoculated with dental plaque obtained from Macaca fascicularis produced methane when the medium contained ethanol, methanol, lactate, acetate or a hydrogen + CO2 atmosphere. Methane formation in the enrichments was inhibited by oxidation of the culture medium, autoclaving or the addition of 2-bromoethane sulfonic acid (BES). The methane producing enrichments were observed to contain fluorescent cocci occurring singly and in short chains. It was concluded that methane formation in the monkey dental plaque was the result of the presence of methanogenic bacteria.     

Modification of Lignins by Growing Cells of the Sulfate-Reducing Anaerobe Desulfovibrio desulfuricans

The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans was grown on medium supplemented with either Kraft lignin or lignosulfonate. Only lignosulfonate contributed to the growth of D. desulfuricans cells, by replacing sulfate, a natural electron acceptor for this microorganism. Kraft lignin added to the culture medium could not substitute for lactate or sulfate, both necessary culture medium components. However, it was found to enhance the viability of D. desulfuricans cells. When changes occurring in lignin during growth of Desulfovibrio cultures were monitored, it was found that both lignin preparations could be partially depolymerized. Spectrophotometric and elemental analysis of biologically treated lignins suggested that both the polyphenolic backbone and lignin functional groups were affected by D. desulfuricans. After treatment, a twofold increase in the sulfur content of Kraft lignin and a minor decrease (14%) in the sulfur content of lignosulfonate were observed. After biological treatment, Kraft lignin and lignosulfonate both bound larger quantities of heavy metals.     

Endogenous levels of abscisic acid and indole-3-acetic acid during in vitro rooting of Wild Cherry explants produced by micropropagation

Levels of endogenous ABA and IAA were quantified during the first week of in vitro rooting of Wild Cherry (Prunus avium L.) using IBA in the culture medium. Hormones were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme linked immunosorbent assay (ELISA), after a purification of the methanolic extracts by high-performance liquid chromatography (HPLC).Root primordia started to differentiate from day 5 at the basal part of the explants. ABA and IAA showed considerable changes and high levels were detected during the first week of culture. ABA levels increased transiently mainly in the apical part during root formation. Exogenous IBA was possibly transformed into IAA mainly in the basal part of the explants.     

Production of peroxidase with horseradish hairy root cells in a two step culture system

Horseradish hairy root cells transformed by a soil bacterium Agrobacterium rhizogenes had peroxidase (POD) activity comparable to that of the original plant root tubers. To enhance POD production by the hairy root culture, various additives to the medium were tested including casein hydrolysates and plant extracts. Polypepton addition had a significant effect on the growth and POD production; at low concentrations (below 1 g/l) the growth was stimulated, while at high concentrations (3–10 g/l), POD activity in the cells was enhanced in spite of a low growth rate. Therefore, the hairy roots were at first cultured in the medium with 1 g/l Polypepton, and then 5 g/l Polypepton was added to enhance intracellular POD activity. POD activity in this two step culture system was 5.4 times higher than that in conventional culture in Polypepton-free medium.     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal

Summary Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to the availability of glucose and fructose following heat-induced hydrolysis of sucrose and the AC-mediated adsorption of inhibitory compounds produced during autoclaving.     

Effect of activated charcoal and pruning of the taproot on the in vitro mycorrhization of Hevea brasiliensis Müll. Arg.

In vitro mycorrhization of Hevea brasiliensis under autotrophic culture conditions is a promising methodology to produce plantlets adapted to overcome stresses during acclimatization. However, to succeed in the in vitro production of mycorrhizal plantlets, root production and subsequent colonization by the mycorrhizal fungus need to be optimized. Plantlets of H. brasiliensis clone PB 260 were grown in contact with the extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833. Addition of activated charcoal to the medium and pruning of the taproot were evaluated for their effects on root growth and colonization. None of the treatments stimulated the early formation of new roots. However, total root length, total root colonization, and production of arbuscules and intraradical spores/vesicles were significantly higher in plantlets grown in the presence of activated charcoal (especially after 13 wk of culture). In contrast, total root colonization was significantly lower in the pruned plantlets, while total root length and arbuscule formation were not affected. None of the treatments affected activities of succinate dehydrogenase and alkaline phosphatase measured in the extraradical mycelium of the fungus. It appeared that the addition of activated charcoal to the culture medium favored root growth and mycorrhization of rubber plantlets under in vitro culture conditions, while taproot pruning did not favor these parameters.     

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

The utilisation and accumulation of ¹⁵N-labeled metabolites by a ¹⁵N-labeled transformed root culture of Daturastramonium L. was investigated by in vivo ¹⁵N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [¹⁵N]glutamate and [¹⁵N]glutamine were rapidly depleted and there was an increase in the ¹⁵N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of ¹⁵N-label in the hydroxy-cinnamoylputrescines was reduced when the de-differentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing α-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-differentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, dl-α-difluoromethylarginine and dl-α-difluoromethylornithine. In-vivo ¹⁵N-NMR spectra of roots grown in ¹⁵N-enriched medium supplemented with these inhibitors showed that the ¹⁵N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. dl-α-difluoromethylarginine also prevented the phytohormone induced de-differentiation of the root cultures, and this effect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-differentiation of the root culture. Received: 13 September 1997 / Accepted: 12 November 1997     

Micropropagation and in vitro flowering of the bamboo Dendrocalamus hamiltonii Munro

Nicotinic acid, pyridoxine, and picloram were stable in a liquid MS culture medium (pH 5.5–5.6) during autoclaving and during cell-free incubation in the dark at 5°C or 25°C for up to 6 weeks. Thiamine loss under the same conditions was 16% at 5°C and 18% at 25°C. Five percent of the sucrose in the liquid medium was hydrolyzed during autoclaving. During cell-free incubation in the light (100 E m^–2 s^–1) at 25°C, pyridoxine was not detected after 6 days, while 78% of the picloram and 56% of the thiamine were degraded after 6 weeks. All of the niacin and pyridoxine, 13% of the picloram and 42% of the thiamine in a liquid MS culture medium were utilized in 4 days by potato (cv. Lemhi Russet) tuber suspension cultures growing in the dark at 25°C.Abbreviations BS Gamborg et al. medium (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - PAA phenylacetic acid     

Effects of precursors on serially propagated Digitalis lanata leaf and root cultures

Serially propagated Digitalis lanata leaf and root cultures established from germinated seeds were studied for digoxin production. Leaf cultures were grown and maintained in a medium containing benzyl adenine, and root cultures in the same medium with indoleacetic acid. A consistently high digoxin content, as determined by radio-immunoassay, is present in the leaf culture (9.0 mg % dry wt) and in root culture (1.9 mg % dry wt) as compared to unorganized cells (0.06 mg % dry wt). Leaf liquid cultures grew very rapidly as compared to the root cultures and the unorganized cell suspension cultures. The concentration of digoxin increased in both leaf and root cultures by adding to the medium either sodium glycocholate, cholesteryl acetate, or progesterone. Smilageninacetate increased the digoxin content of root cultures but not that of leaf cultures. Lanosterol and 5β-androstan-3,17-dione did not significantly increase the concentration of digoxin. Deoxycholic acid was toxic to the tissues studied.     

Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection

Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis index.     

Biotransformation of cinnamyl alcohol to rosavins by non-transformed wild type and hairy root cultures of Rhodiola kirilowii

Non-transformed wild type (NTWT) and hairy root cultures of Rhodiola kirilowii were grown in medium supplemented with 2.5 mM cinnamyl alcohol as a precursor and/or sucrose (1 %) on the day of inoculation or on the 14th day of culture. Rosarin, rosavin, and rosin were produced by NTWT root culture but only rosarin and rosavin by hairy roots. Approximately 80 and 95 % of the glycosides were released into the medium for NTWT and hairy root cultures, respectively. The highest rosavin yield, 505 ± 106 mg/l, was in hairy root culture when cinnamyl alcohol was applied on the day of inoculation with the addition of sucrose on the 14th day of culture. For rosin production, supplementation with cinnamyl alcohol alone on day 14 was more favourable with the highest amount 74 ± 10 mg/l in NTWT root culture. Only traces of rosarin were detected.     

Tetanus Toxin Production in the Absence of Protein

A modification of the Mueller medium for tetanus toxin production is presented, on which an adapted strain produces high yields of toxin (70 to 90 flocculating units per ml) in the absence of animal protein extracts.

The range of iron concentration in the medium was established within which the toxigenic activity of the culture can be made to vary, and the mathematical nature of the variation is presented. Darkening of cultures during incubation indicates departure from optimal conditions. Heat input to the medium during autoclaving, originally undertaken solely for purposes of sterilization, is an important physicochemical factor in the toxigenicity of the culture.

     

Association of Azospirillum with Grass Roots

The association between grass roots and Azospirillum brasilense Sp 7 was investigated by the Fahraeus slide technique, using nitrogen-free medium. Young inoculated roots of pearl millet and guinea grass produced more mucilaginous sheath (mucigel), root hairs, and lateral roots than did uninoculated sterile controls. The bacteria were found within the mucigel that accumulated on the root cap and along the root axes. Adherent bacteria were associated with granular material on root hairs and fibrillar material on undifferentiated epidermal cells. Significantly fewer numbers of azospirilla attached to millet root hairs when the roots were grown in culture medium supplemented with 5 mM potassium nitrate. Under these growth conditions, bacterial attachment to undifferentiated epidermal cells was unaffected. Aseptically collected root exudate from pearl millet contained substances which bound to azospirilla and promoted their adsorption to the root hairs. This activity was associated with nondialyzable and proteasesensitive substances in root exudate. Millet root hairs adsorbed azospirilla in significantly higher numbers than cells of Rhizobium, Pseudomonas, Azotobacter, Klebsiella, or Escherichia. Pectolytic activities, including pectin transeliminase and endopolygalacturonase, were detected in pure cultures of A. brasilense when this species was grown in a medium containing pectin. These studies describe colonization of grass root surfaces by A. brasilense and provide a possible explanation for the limited colonization of intercellular spaces of the outer root cortex.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.