The Effect of a Submersion Pretreatment on In Vitro Explant Growth and Shoot Regeneration from Hypocotyls of Flax (Linum Usitatissimum)

This study was carried out to determine the effect of temporary submersion of hypocotyl segments in water on in vitro explant growth and shoot regeneration on MS (Murashige and Skoog, 1962) medium supplemented with 1 mg l⁻¹ BAP (6-benzylaminopurine) and 0.02 mg l⁻¹ NAA (naphthaleneacetic acid) in three flax cultivars. It was observed that water-treated hypocotyl explants gave rise to the highest values with respect to shoot regeneration percentage, shoot number per hypocotyl, shoot length and total shoot number per Petri dish, successful rooting and plantlet establishment. This procedure may be applicable for other species cultured in vitro.     

The effect of bacterial contamination on the growth and gas evolution ofIn vitro cultured apricot shoots

Summary Shoots of “San Castrese” and “Portici” apricots (Prunus armeniaca L.) free of cultivable bacteria, shoots of the same origin exhibiting bacterial contamination after repeated subcultures, and contaminated shoots treated with cefotaxime were compared for gas exchange, proliferation rate, and fresh and dry weight. Cultures of San Castrese contaminated byBacillus circulans andSphingomonas paucimobilis, and of Portici contaminated withStaphylococcus hominis andMicrococcus kristinae, including those treated with cefotaxime, showed comparable shoot weights and lower proliferation rates than healthy cultures. Bacteria, even if not visible until the end of subculture, markedly influenced the gaseous composition of the jar headspace. Healthy cultures clearly showed photosynthetic activity at 60 μM·m⁻²·s⁻¹ photosynthetically active radiation; in contrast, oxygen quickly decreased and carbon dioxide increased in contaminated cultures, including those treated with cefotaxime, in which bacteria became visible in the culture medium only after repeated subcultures.     

The in Vitro Growth of Human Chondrocytes

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20–72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients.     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis, starch and total sugar accumulation, in kiwifruit culturedin vitro

Shootlets of kiwifruit plants (Actinidia deliciosa) were culturedin vitro. Combinations of light intensity, Mg and sucrose in the cultures showed that an increase of light intensity resulted in a corresponding increase of the relative size of the leaf mesophyll cells and in a decrease of the numbers of chloroplasts and contained starch grains. The addition of sucrose to the substrate media negatively affected the size of the mesophyll cells under normal Mg concentration (35 mg l⁻¹), and positively under high Mg concentration (105 mg l⁻¹ ). Sucrose further resulted in an increase in the numbers of chloroplasts and contained starch grains. The photosynthetic capacity of leaves greatly increased when Mg concentration was enhanced and sucrose was excluded from the nutrient substrate. Total sugar accumulation in all treatments was favoured by normal light intensity and addition of sucrose.     

A comprehensive review on nettle effect and efficacy profiles, Part I: Herba urticae

Nettle herb is recommended for complaints associated with rheumatoid arthritis, osteoarthritis and urinary tract infections. We therefore conducted a comprehensive review of the literature to summarize the pharmacological and clinical effects of this plant material. Although clinical and experimental studies suggest that nettle herb has some anti-inflammatory properties, clinical evidence beyond doubt is lacking. Nettle preparations exert a number of promising in vitro and in vivo effects, however, further studies are needed to support these results and to find out if these effects are surrogates for clinical relevant effects in humans.     

Embryonic Chicken Trachea as a New In Vitro Model for the Investigation of Mucociliary Particle Clearance in the Airways

Mucociliary clearance (MC) is an important defense mechanism of the respiratory system to eliminate inhaled and possibly noxious particles from the lung. Although the principal mechanics of MC seem to be relatively clear there are still open questions regarding the long-term clearance of particles. Therefore, we have developed a new set-up based on embryonic chicken trachea (ECT) to investigate mucociliary particle clearance in more detail. ECT was placed in an incubation chamber after carbon particles were applied and tracked using optical microscopy. The aim of the study was to validate this model by investigating the impact of temperature, humidity and drugs on particle transport rates. Particles were transported reproducibly along the trachea and clearance velocity (2.39 ± 0.25) mm/min was found to be in accordance to data reported in literature. Variation in temperature resulted in significantly reduced MC: (0.40 ± 0.12) mm/min (20 °C); (0.42 ± 0.10) mm/min (45 °C). Decreasing humidity (99–60%) had no significant effect on MC, whereas reduction to 20% humidity showed a significant influence on particle clearance. The use of different cilio- and muco-active drugs (Propranolol, Terbutalin, N-acetylcysteine) resulted in altered MC according to the pharmacological effect of the substances: a concentration dependent decrease of MC was found for Propranolol. From our results we conclude that this model can be employed to investigate MC of particles in more detail. Hence, the model may help to understand and identify decisive physico-chemical parameters for MC and to answer open questions regarding the long-term clearance phenomenon. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

濒危植物盘龙参种子的非共生萌发及种苗的快繁研究

以盘龙参种子为材料,筛选离体条件下适宜种子非共生萌发、种苗快繁的培养基。结果表明:盘龙参种子在无植物生长物质的培养基中能够萌发,但不能发育成苗;在含有1.0mg·L-1 KT、0.1mg·L-1 IAA和0.1mg·L-1 GA3的培养基中萌发,并能进一步发育形成种苗;在较高浓度生长素(1.2mg·L-1 NAA)的培养基中不能萌发。种苗在较高浓度细胞分裂素和较低浓度生长素配比的培养基中能够增殖,最高增殖系数可达到2.8,转入壮苗生长培养基中培养80d后可以移栽温室。最适增殖培养基为1/2MS+12.0mg·L-1 6-BA+0.1mg·L-1 NAA+10.0mg·L-1腺嘌呤;最适壮苗生根培养基为1/2MS+1.0mg·L-1 KT+0.1mg·L-1 IAA+10.0mg·L-1腺嘌呤。     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

Specific Features of Development of Megagametophytes and Embryos of the Siberian Stone Pine in vitro

Seedlings were grown in vitro from fertilized eggs and immature embryos of the Siberian stone pine. Cultivation of megagametophytes on a hormone-containing Murashige-Skoog medium from the egg formation until the globular embryo stage made it possible to manipulate fertilization and embryogenesis. Immature embryos are the most promising for in vitrocultivation. Their maturation and germination proceed within seven days of cultivation. When zygotic embryos were cultivated, adventitious buds were formed from cells at the cotyledon base and tips. When adventitious buds were subcultivated on a medium containing benzylaminopurine and naphthylacetic acid, organogenic callus and shoots were formed. Thus, cultivation of megagametophytes and embryos of the Siberian stone pine led to the completion of embryogenesis and formation of viable of seedlings.     

Micropropagation of the endangered <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis> Baijnath

Summary The species, Kniphofia leucocephala is extant at only one location, Langepan, KwaZulu-Natal in South Africa, where the population is threatened by afforestation and possibly grazing. Consequently, a continuous culture system was established as part of a program for the propagation and re-introduction of plants into the wild. The efficiency of the system in terms of shoot multiplication and, particularly, the frequency and rate of root initiation was strongly influenced by the concentration of benzyladenine in the shoot multiplication medium. The optimum shoot multiplication medium for subsequent root initiation contained 2 mgl⁻¹ (8.9 μM) benzyladenine alone. The shoots were successfully rooted and acclimatized. Approximately 200 shoots can be produced from one shoot after five 4-wk cycles. Thus, large numbers of plantlets can be propagated in this continuous culture system, serving conservation interests.     

Autotrophic denitrification and inhibitory effect caused by the injection of spent sulfidic caustic in a modified Ludzack-Ettinger process

As spent sulfidic caustic (SSC) from petroleum plants contains a high concentration of alkalinity and sulfur compounds, SSC can be applied in sewage treatment system as an electron donor for autotrophic denitrification. In our previous study, the reuse of SSC in the biological nitrogen process was successful, and some neutralization may be required for stable treatment performance. In this study, the pH of SSC was neutralized to 12.0 from 13.3, and the modified Ludzack-Ettinger process was conducted for 90 days with the municipal wastewater. Some toxic effects of SSC on microorganisms were tested via a specific oxygen uptake rate (SOUR) assay. According to the SOUR assay, as compared with no SSC injection condition, SOUR was reduced by approximately 5.4% when 4 mL SSC/L was injected and the effective concentration of a toxicant causing 50% inhibition of the microorganism’s activity (EC₅₀) was 22.6 mL/L. During the days of operation, the COD removal and nitrification efficiency were over 53.0 and 98.2%, respectively. The TN removal efficiency was 56.6% and the nitrogen removal rate (NRR) was 0.15 kg/m³·d when the hydraulic retention time (HRT) in the anoxic tank was 3 h. The ratio of nitrifying bacteria was unaffected by the HRT, and Nitrobacter spp. and Nitrospira genus existed at similar ratios. The ratio of T. denitrificans increased after the injection of SSC and was approximately 6.5%.     

Restorative Morphogenesis in vitro in the Annual Medic in the Presence of Benzylaminopurine

Four annual medic species (Medicago orbicularis (L.) All., M. rigidula (L.) Desr., M. scutellata (L.) Miller, and M. rugosa Desr.) were used as model objects for studying the spectrum of morphogenetic reactions in vitro. The seeds were incubated on nutrient media with benzylaminopurine at different concentrations until germination and, thereafter, the explants of seedlings were cultivated in order to obtain primary calluses and morphogenetic structures. Normal and abnormal (with reduced root and/or apex) seedlings were cultivated in the presence of benzylaminopurine. Further cultivation of explants from the seedlings of both types showed a considerable intra- and interspecific polymorphism by the capacity for callusogenesis, frequency of primary restorative reactions, and pattern of microreproduction in vitro. In the control (the seeds were incubated on a hormone-free medium), no cases of microreproduction by way of organogenesis or somatic embryogenesis were observed. In all experimental variants, the restorative reactions preceded microreproduction in vitro.     

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl^-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 M was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 m) using an IBA powder dip (3 g IBA kg^-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige and Skoog medium     

Effect of Some Antibiotics on the In Vitro Morphogenetic Response from Callus Cultures of Coryphantha Elephantidens

The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm^–3 kanamycin, which was under study.     

In vitro cytotoxicity of heavy metals,acrylamide, and organotin salts to neural cells and fibroblasts

The cytotoxicity of neurotoxic agents was determined for a series of brain-derived cell types and compared with their toxic effects on BALB/c 3T3 fibroblasts, using the neutral red assay. Ranking of toxicants according to their potencies was the same for all cells tested and was in the order of methylmercury>cadmium> mercury>zinc>acrylamide. For a series of di- and triorganotins the ranking order was dibutyl>diphenyl>dibenzyl>diproyl> diethyl>dimethyltin and triphenyl>tribenzyl>trimethyltin, respectively. The test was sensitive enough to detect structure activity relationships between the degree of toxicity and the hydrophobic characteristics of the agents tested.Abbreviations Be₂Sn dibenzyltin dichloride - Be₃Sn triphenyltin dichloride - Bu₂Sn dibutyltin dichloride - CdCl₂ cadmium chloride - CH₃HgCl methylmercuric chloride - DMEM Dulbecco's Modified Eagle's Medium - Et₂Sn diethyltin dichloride - FBS fetal bovine serum - HgCl₂ mercuric chloride - Me₂Sn dimethyltin dichloride - Me₃Sn trimethyltin hydroxide - Phe₂Sn diphenyltin dichloride - Phe₃Sn triphenyltin hydroxide - Pr₂Sn dipropyltin dichloride - ZnCl₂ zinc chloride     

Use of an isolated guinea-pig ileum assay to detect bioactive compounds in microalgal cultures

An isolated guinea-pig ileum preparation was used to screen for bioactive compounds from algae. 212 culture supernatants and methanolic extracts of randomly chosen marine and freshwater algae were tested for their effect on electrically evoked muscle contractions (recorded as a change in tension) and on the resting muscle tone. 15 out of 42 (35%) of the marine algae tested and 5 out of 64 (8%) of freshwater algae gave positive results. Of the 20 algae giving positive results, 6 had previously been shown to produce bioactive compounds (mainly toxins) but we can find no reports in the literature of bioactive compounds from the remaining 14. Of these 14 cultures, 9 were axenic and therefore production of the biological activity can be assigned unambiguously to the alga. These results confirm the usefulness of the guinea-pig ileum preparation as a screen for bioactive compounds from microbial cultures.     

Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka

Summary Phytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.