Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

Relationships of endogenous plant hormones to accumulation of grain protein and starch in winter wheat under different post-anthesis soil water statusses

Accumulation of protein and starch in grain is a key process determining grain yield and quality in wheat. Under drought or waterlogging, endogenous plant hormone levels will change and may have an impact on the yield and quality of wheat. In a greenhouse experiment, four winter wheat (Triticum aestivum L.) varieties differing in grain protein content, Heimai 76, Wanmai 38, Yangmai 10 and Yangmai 9, were subjected to drought (SRWC = 4550%, DR), waterlogging (WL) and moderate water supply (SRWC = 7580%, CK), beginning from 4 days post-anthesis (DPA) to maturity. On the 10 (grain enlargement stage) and 20 (grain filling stage) DPA, endogenous abscisic acid (ABA), gibberellins (GA₁₊₃), indole-3-acetic acid (IAA) and zeatin riboside (ZR) were determined in sink and source organs of wheat plants by enzyme linked immunosorbent assay (ELISA). The patterns of hormonal changes were similar in four varieties. The ABA levels were much higher under DR and WL than under CK. Compared with CK, GA₁₊₃ levels in whole-plant under DR and WL changed a little at 10 DPA, but markedly decreased under DR and WL at 20 DPA. Changes of endogenous IAA level under DR and WL exhibited a complicated pattern, depending on organs and growth stages. Particularly at the 20 DPA, the mean levels of IAA in roots, leaves and grains decreased significantly under DR and WL. In comparison with CK, ZR levels in all organs significantly decreased under DR and WL at both stages. The correlation analyses between yields and contents of starch and protein in grains and levels and ratios of four hormones in source and sink organs indicated that the changes in yield and content of grain starch and protein under DR and WL were associated with the reduced IAA, ZR and GA₁₊₃ levels and elevated ABA level in plants, especially in grains. It was proposed that the changed levels of endogenous hormones under post-anthesis DR and WL might indirectly affect protein and starch accumulation in grains by influencing the regulatory enzymes and processes.     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes

In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes. Leucocytes were preincubated for 30min with different concentrations of sugar (alpha-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays. The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry. The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent. More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked. Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed. The results demonstrated that the phagocytosis of yeast was completely blocked by this substance. The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes.     

Effects of different stressor agents on gilthead seabream natural cytotoxic activity

Several common situations in gilthead seabream (Sparus aurata L.) farming, such as air exposure, crowding and the use of anaesthetics, have been demonstrated to be stressful. In the present study, these conditions were simulated in the laboratory, after which head-kidney natural cytotoxic cell (NCC) activity was evaluated. For this, several specimens were air exposed for 2 min, returned to the aquarium and sampled from 0 to 4 days after exposure. NCC activity was significantly lower on the day following air-exposure compared with the control (rested fish) but not at any other time studied. Other fish were crowded (100 kg biomass m(-3), 2 h), returned to an aquarium with the same density as the control group (9 kg m(-3)) and sampled from 0 to 4 days after treatment. Head-kidney NCC activity was statistically increased compared with the control (resting) fish, 1 day after crowding. Anaesthesis for 1 h with 60 or 200 microl 2-phenoxyethanol l(-1)had no significant effect on NCC activity, while the use of 50 mg MS222 l(-1)for 1 h reduced such activity (by about 40%) compared with the control. In other experiments, fish were consecutively treated with crowding and anaesthetics. When treated with the lowest 2-phenoxyethanol concentration after crowding, the NCC activity inhibition was abolished compared with the activity in fish treated either with crowding or anaesthetic alone, while the use of the highest concentration increased such inhibition. The use of MS222 after crowding did not produce any differential effect compared with the fish treated with only one of the factors. In conclusion, NCC activity is affected differently according to the stress factor applied (hypoxia, crowding and/or anaesthetics). Differences in the effects provoked by these stressors on other seabream innate immune parameters are discussed.     

Production,storability, and regeneration of shoot tips of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) encapsulated in calcium alginate hollow beads

Summary Shoot tips, of four potato cultivars (Désirée, Genet, Tigoni, and Tomensa), 3–4 mm in size, were precultured for 2 d on Murashige and Skoog (MS) solid medium, then encapsulated in calcium alginate to produce hollow bead synthetic seed capsules averaging 0.78 cm in diameter. Regeneration and ‘regrowth’ were tested on MS solid medium and on soil in the greenhouse, respectively. The encapsulated shoot tips were stored at 4 and 10°C for up to 390 d. For all cultivars, the encapsulated shoot tips stored at both temperatures for 180 d and at 4°C,for 270 d, 100% regeneration on MS solid medium was recorded. After 360 d in storage at 4°C, 70.8% (Tigoni), 66.7% (Genet), 58.3% (Désirée), and 51.5% (Tomensa) regeneration was recorded on MS medium, reducing to 15% (Tigoni), 25% (Genet), 10% (Désirée), and 0% (Tomensa) regeneration after 390 d in storage. ‘Regrowth’ of 93–100% was recorded for non-stored encapsulated shoot tips, directly transferred on soil in the greenhouse after a 2 wk preculture on MS solid medium with an added fungicide (carbendazim) in the encapsulating gel. The ‘regrown’ shoot tips produced plants showing normal development. The results presented here demonstrate that hollow bead synthetic seed capsules are an alternative propagating method for potato seed production.     

In vivo and in vitro effects of cadmium on H+ ATPase activity of plasma membrane vesicles from oat (Avena sativa L.) roots

The effect of an in vivo and in vitro treatment with cadmium on transport activities of root plasma membrane enriched vesicles was studied in oat (Avena sativa L. cv. Argentina) plants. Addition of 100 mumol/L CdSO4 to nutrient solution decreases both proton transport activity and ATPase activity to the same level. In vitro experiments show that cadmium seems to have a differential inhibiting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H(+)-accumulation, suggesting that cadmium would interfere with membrane permeability properties. This is indeed the case. The results demonstrate that cadmium decreases passive permeability to protons.     

Age-dependent response of maize leaf segments to cadmium treatment: effect on chlorophyll fluorescence and phytochelatin accumulation

The relationship between the age of leaf tissue and response of the photosynthetic apparatus and phytochelatin accumulation to Cd treatment was studied. Studies were carried out with seedlings of Zea mays L. cv. Hidosil grown in the presence of 100-200 mumol/L Cd for 14 days under low light conditions. The third leaf was divided into 3 segments of equal length differing in the stage of tissue maturity and used for measurements of chlorophyll content, chlorophyll fluorescence, glutathione and phytochelatin content and Cd accumulation. A close relationship between the age of leaf tissue and response of the photosynthetic apparatus to Cd was shown. Cadmium (200 mumol/L) reduced photochemical processes more in older than younger leaf segments as seen in the Chl fluorescence parameters Fv/F0, and t1/2, while the chlorophyll fluorescence decrease ratio (Rfd) was inhibited more strongly in younger ones. Fv/Fm was slightly affected. Cd-induced enhancement of GSH content was correlated with higher phytochelatin accumulation to a greater extent in younger than in older leaf segments. Phytochelatin level corresponded to changes of photochemical processes in older leaves. The peptide thiol:Cd molar ratio for the phytochelatins varied depending on Cd concentration and age of leaf segments. The protective role of phytochelatins for the photosynthetic apparatus is discussed.     

Coumarin inhibits the growth of carrot (Daucus carota L. cv. Saint Valery) cells in suspension culture

We used a carrot (Daucus carota L. cv. Saint Valery) cell suspension culture as a simplified model system to study the effects of the allelochemical compound coumarin (1,2 benzopyrone) on cell growth and utilisation of exogenous nitrate, ammonium and carbohydrates. Exposure to micromolar levels of coumarin caused severe inhibition of cell growth starting from the second day of culture onwards. At the same time, the presence of 50 mumol/L coumarin caused accumulation of free amino acids and of ammonium in the cultured cells, and stimulated their glutamine synthetase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxylase activities. Malate dehydrogenase, on the other hand, was inhibited under the same conditions. These effects were interpreted in terms of the stimulation of protein catabolism and/or interference with protein biosynthesis induced by coumarin. This could have led to a series of compensatory changes in the activities of enzymes linking nitrogen and carbon metabolism. Because coumarin seemed to abolish the exponential phase and to accelerate the onset of the stationary phase of cell growth, we hypothesise that such allelochemical compounds may act in nature as an inhibitor of the cell cycle and/or as a senescence-promoting substance.