Effects of TRH on cell proliferation of rat pituitary cells (GH3)

Summary Chronic treatment (more than 3 d) of GH₃ cells, cloned rat pituitary cells producing prolactin, with 100 nM TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e. insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity (MSA) instead of serum. TRH stimulated prolactin production by GH₃ cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 nM, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 nM, it inhibited the growth significantly. In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on the growth of GH₃ cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five factors other than MSA. In conclusion, TRH could inhibit cell growth of GH₃ in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium.     

Effects of the medium ammonium-nitrate ratio on competence for asparagus cell division induced by phytosulfokine-α

The effects of medium ammonium-nitrate ratio on cell proliferation were investigated using a low cell-density culture of Asparagus officinalis L., which was triggered by a peptidal plant growth factor, phytosulfokine-α (PSK-α). The asparagus cells proliferated the most in a medium with an ammonium-nitrate ratio of 0:30 mM and could be maintained without significant loss of responsiveness to PSK-α for at least 96 h from the beginning of culture. In this medium, single cells gave rise to microcalli at initial densities as low as 3.2×10² cells/ml as long as PSK-α was present in the medium. Increasing the ammonium-nitrate ratio resulted in severe inhibition of cell proliferation at a low cell density, even if PSK-α was added to the medium. Received: 6 May 1997 / Revision received: 5 August 1997 / Accepted: 2 September 1997     

Mitochondrial genome structure of a cytoplasmic hybrid between tomato and wild potato

A physical map of the mitochondrial genome was constructed for a male-sterile tomato, MSA1, which had been generated by an asymmetric cell fusion between tomato (Lycopersicon esculentum) and wild potato (Solanum acaule). The entire genomic sequence of the MSA1 mitochondria (450 kb) was represented by five maps. Even if sequence duplications were taken into consideration, at least two linkage groups (maps 1–4 and map 5) were necessary to show the overall genome. The mitochondrial genome structure of MSA1 was also analyzed by comparing the Southern hybridization patterns of MSA1 and its parents (tomato and wild potato). The mitochondrial genome of MSA1 consists of a complex mixture of the parental genomes with at least 11 molecular recombination events. Received: 23 February 1998 / Revision received: 2 March 1998 / Accepted: 14 March 1998     

Growth and rosmarinic acid production in cell suspension cultures of Salvia officinalis L.

Rosmarinic acid (RA) is a natural antioxidant produced by cell suspension cultures of sage (Salvia officinalis L.). The growth and production of RA by these cells can be modified by the type of culture medium. Production can be increased 10-fold to attain 6.4 g.1^-1 under optimal conditions. Investigation of kinetics showed that a change in the medium caused shifts in peaks of growth and production, and modifications of the cell metabolism. RA production can be correlated with growth or begins only when growth has stopped.     

Capacity for aluminium uptake depends on brefeldin A-sensitive membrane traffic in tobacco (Nicotiana tabacum L. cv. BY-2) cells

The relationship between cellular growth and aluminium (Al) uptake was examined by applying brefeldin A, a vesicle transport inhibitor, to cultured tobacco (Nicotiana tabacum L. cv. BY-2) cells. Cultured cells almost completely lost the capacity for Al uptake when pre-incubated for 1–3 h in a minimal medium. Pre-incubation also diminished subsequent growth in a culture medium. However, competency for Al uptake (20 μm, pH 4.5) was sustained in a dose-dependent and reversible manner when cells were treated with brefeldin A (10 μm), an inhibitor of Golgi-mediated secretion, prior to and during the incubation in minimal medium. Received: 24 September 1998 / Revision received: 24 November 1998 / Accepted: 5 December 1998     

Nutrient and salt relations of <Emphasis Type="Italic">Pterocarpus officinalis</Emphasis> L. in coastal wetlands of the Caribbean: assessment through leaf and soil analyses

Pterocarpus officinalis L. is a dominant tree of freshwater coastal wetlands in the Caribbean and the Guiana regions. It is frequently associated with mangroves in areas with high rainfall and/or surface run-off. We hypothesized that P. officinalis is a freshwater swamp species that when occurring in association with mangroves occupies low-salinity soil microsites, or alternatively that it possesses mechanisms preventing accumulation of salt in photosynthetic tissues. To test this we compared the mineral composition of soils and leaves of several species in two Pterocarpus forests in Puerto Rico associated with coastal mangroves, Sabana Seca and Punta Viento. Results indicate that (1) Sabana Seca has low soil salinity values even in the P. officinalis and Laguncularia racemosa mixed zone. In Punta Viento, salinity in the mixed zone was higher than in the Pterocarpus forest, but much lower than in the mangrove area; (2) In both forests, leaves of P. officinalis showed much lower Na concentrations than mangrove species. The K/Na ratios were 16–20 times higher in P. officinalis, indicating preferential absorption of K against Na. The mangrove fern (Acrostichum aureum) growing side by side with P. officinalis and L. racemosa in Punta Viento also revealed high Na exclusion capacity. We found an asymmetric distribution of cations in the blade and rachis of the P. officinalis compound leaves in both sites. The rachis accumulates more Na and Ca, but less Mg than the leaf blade. This sequestration of Na in the rachis prevents salt damage of photosynthetic tissue in the leaf blade.     

Excised rootstock roots cultured in vitro

Root cultures have been established successfully for the Prunus rootstocks Adafuel and Adarcias (Prunus×amygdalopersica), A843 (P. armeniaca), Mariana 2624 (P. cerasifera×munsoniana) and Myrobalan 605AD (P. cerasifera), and for the apple rootstock Jork 9 (Malus×domestica). High percentages of root tips grew during the first 15 days and then decreased. Root growth was affected by culture conditions and the composition of the culture medium. Liquid medium was preferable as increasing agar concentrations reduced root growth. Darkness, instead of a 16-h photoperiod, was beneficial for Adafuel, but not the other genotypes. Sucrose at 3% was better than higher concentrations. Full Murashige and Skoog salts sustained better root growth than dilutions to 1/2 or 1/4. The addition of various organic supplements such as coconut water, casein hydrolysate or malt extract did not improve root growth, and sucrose was the best carbon source tested. Data presented here support the notion that excised root culture is an efficient experimental model to study the response to various factors, since controlled variations in the culture medium, such as those studied here, had a very noticeable effect on root length. Received: 4 June 1997 / Revision received: 3 February 1998 / Accepted: 1 March 1998     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.     

Nutritional Composition Profiles and Quality Evaluation of Different Cultivars of Asparagus Officinalis with Potential as Functional Foods and Health-Care Products

Asparagus officinalis is a health-care vegetable with homology value of medicine and food. The quality of A. officinalis is greatly different from various cultivars. It is essential to reveal the relationship between the variety and quality. This study investigated six nutritional compositions in ten A. officinalis cultivars, including amino acid, mineral substance, carbohydrate, vitamin C, protein and total sugar. Five chemometrics methods were further employed to evaluate their quality. The results consistently showed that ten varieties were divided into three grades as nutritional composition differences. HuaMiaoF1, JinGuan and FeiCuiMingZhu were grouped into cluster3 with the best quality, and Atlas and Jersey Giant were grouped into cluster1 with the lowest quality. Therefore, HuaMiaoF1, JinGuan and FeiCuiMingZhu can be suggested as good raw materials for medicine, food and health-care products industries. Meanwhile, the comprehensive application of five chemometrics methods was confirmed as a reliable methodology for quality evaluation of A. officinalis.     

Neuroprotective Effect of <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis> Extract on Human Dopaminergic Cell line,SH-SY5Y

Hydrogen peroxide (H₂O₂) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson’s disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H₂O₂-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H₂O₂-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H₂O₂. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H₂O₂-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

陕南秦巴山区厚朴群落土壤肥力评价

以秦巴山区7个厚朴群落为对象,分析土壤养分含量的变化规律,同时对各群落土壤肥力进行评价,以期为该区土壤肥力的提高和厚朴群落配置模式的筛选提供理论依据和实践指导。结果表明,秦巴山区厚朴群落间土壤养分含量差异显著;除全磷和全钾外,随土层深度的增加,土壤有机质、全氮、碱解氮、速效磷和速效钾含量逐渐减小。厚朴群落土壤肥力综合指数从大到小依次为:4a凹叶厚朴(1.054)2a凹叶厚朴(0.882)山茱萸-凹叶厚朴混交林(0.673)7a尖叶厚朴(-0.243)7a凹叶厚朴(-0.713)杜仲-凹叶厚朴(-0.812)11a凹叶厚朴(-0.840)。因此随着林龄的增大,厚朴群落土壤肥力有下降的趋势。结合厚朴群落土壤养分差异分析和厚朴群落土壤肥力评价结果,得出山茱萸-凹叶厚朴混交林的土壤肥力相对较好,为该区植被恢复的最佳营造模式。     

Isolation and prominent characteristics of an L-lysine hyperproducing strain of Corynebacterium glutamicum

Summary A strain of Corynebacterium glutamicum was isolated that accumulated up to 44 g/l of L-lysine-HCl from 100 g/l of glucose × H₂O in a simple mineral salts medium. This strain was obtained from the wildtype by two mutagenesis steps. In the first step the aminoethyl-cysteine-resistant strain MH20 was obtained and in the second step the Leu ^— derivative MH20-22B. Enzymatic analysis of the hyperproducer MH20-22B revealed that this strain has feedback-resistant aspartate kinase and is devoid of isopropylmalate dehydratase. In addition, this strain has an extraordinarily high secretion rate of lysine (0.57 mmol/g dry weight and h), whereas strain MH20 has a low secretion rate (0.19 mmol/g per hour), and both strains have comparable cytosolic lysine concentrations. This suggests that the secretory step is influenced n the hyperproducer. Applying gene-directed mutagenesis, the aspartate kinase gene of the isolated strain (coding for feedback-resistant enzyme) was replaced by the gene coding of feedback-sensitive wild-type enzyme. The resulting strain still secretes lysine, although in low amounts (2 g/l). This is proof of the superior role of kinase regulation in metabolite flow and is indicative of unknown mutations, one of which is probably in the secretory system. Correspondence to: L. Eggeling     

Mineral and Carbohydrate Nutrition of Plant Cell and Tissue Cultures

Recent advances in our understanding of the mineral (and carbohydrate) nutrition of cultured plant cells and tissues are reviewed. The methods used for empirical selection of nutrient composition of culture media for different plant genera/species and types of culture are critically evaluated.

The acquisition of nutrients is discussed in terms of their physical availability in the culture medium and uptake from the medium. The effect on uptake of factors such as pH and water potential and the relationship with growth rates and medium depletion are examined in detail.

Finally, some effects of nutrients on morphogenesis of plants are reviewed.     

Plantlet regeneration via somatic embryogenesis in anther callus of Vitis latifolia L.

Anthers of Vitis latifolia L. (wild grape) cultured on Nitsch and Nitsch medium supplemented with 20 μM 2,4-D and 9 μM BAP produced callus after 4–6 weeks. Subculture of callus onto Nitsch and Nitsch medium containing 10 μM NAA produced somatic embryos within 6 weeks. On growth regulator-free Nitsch and Nitsch basal medium somatic embryos converted to plantlets in 6–8 weeks. One gram of callus produced more than 400 somatic embryos with 13.7% being converted to complete plantlets, which were subsequently established in soil. Regenerated plants were found to have mixoploid populations of cells, 2n = 38 and n = 19. Received: 23 May 1998 / Revision received: 21 September 1998 / Accepted: 10 October 1998     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

A method to produce encapsulatable units for synthetic seeds in Asparagus officinalis

A method to produce encapsulatable units for synthetic seeds was developed in Asparagus officinalis L. Encapsulatable units with high conversion ability in non-sterile soil were produced from somatic embryos by a pre-encapsulation culture. The synthetic seeds containing somatic embryos without the pre-encapsulation culture did not germinate in soil. When the pre-encapsulation culture medium did not contain growth regulators, the roots elongated too much to accomplish encapsulation. Several growth regulators were studied and indole-3-acetic acid was considered to be optimum at 28.5 M. The pre-encapsulation culture medium with indole-3-acetic acid inhibited the growth of roots during the pre-encapsulation culture and produced compact encapsulatable units. The growth of roots was promoted when plants were produced from the encapsulatable units. The percent conversion of the synthetic seeds with these encapsulatable units was 72% in non-sterile soil. This is the first report on synthetic seeds in Asparagus officinalis L.     

Co-culture with Daucus carota somatic embryos reveals high 2,4-D uptake and release rates of Arabidopsis thaliana cultured cells

By means of co-culture in growth regulator-free medium we analysed whether factors secreted into the medium of Daucus carota (carrot) somatic embryo cultures would be able to overcome the developmental arrest of globular Arabidopsis thaliana somatic embryos. Instead of Arabidopsis embryogenesis being promoted the development of carrot somatic embryos was inhibited at the globular stage in the presence of Arabidopsis suspension culture aggregates with attached globular embryos. Several experiments showed that this was due to the release of previously accumulated 2,4-D by the Arabidopsis cultures. (1) In addition to arresting carrot embryogenesis, co-culture with Arabidopsis cell suspensions also induced callus formation on Arabidopsis root segments. (2) Both effects only occurred with Arabidopsis suspensions grown in the presence of 2,4-D and not with those grown in the presence of NAA, demonstrating that Arabidopsis is not segregating a “general” inhibiting factor. (3) Both effects could be prevented by either binding 2,4-D to active charcoal or by washing it away by changing the medium daily. (4) Uptake of 2,4-D into Arabidopsis cells during culture in 2,4-D containing medium and subsequent release of 2,4-D after transfer to growth regulator-free medium was measured. (5) These low levels of released 2,4-D (0.2– 0.5 μm) could mimic the observed effects. Taken together these data suggest that the high intracellular 2,4-D content of Arabidopsis cultures may interfere with Arabidopsis somatic embryo development beyond the globular stage. Received: 13 November 1997 / Revision received: 2 February 1998 / Accepted: 16 November 1998