Endogenous gibberellin- and cytokinin-like substances in cultured shoot tissues of apple,Malus pumila cv. Jonathan,in relation to adventitious root formation

The frequencies of adventitious root formation in vitro of isolated shoots from bud cultures of apple (Malus pumila cv. Jonathan) after 1, 7 and 31 subcultures (weeks 5, 29 and 109 after the initial culture) were 5, 78 and 95% respectively. Endogenous gibberellin-like substances (GA) were extracted, chromatographed on SiO₂ partition columns, and assayed on dwarf rice (Oryza sativa cv. Tan-ginbozu). The levels of GA in shoots from the 1st, 7th and 31st subcultures were 40, 19 and 14 ng GA₃ eq./g dry weight of tissue, respectively, a trend which suggests an inverse relationship between endogenous GA level and rooting ability. This is consistent with the fact that applied GA₃ inhibits rooting in apple and many other species. The major peak of GA activity eluted coincidentally with GA₁/GA₃/GA₁₉. Endogenous cytokinin-like substances (CK) were chromatographed on paper and assayed with soybean hypocotyl sections. In contrast to the decrease in GA activity, CK activity increased 1.5–2.7 fold in the later subcultures (cytokinin activity per shoot, however, declined).     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Isolation of tomatine from cultured excised roots and callus tissues of tomato

Tomatine was present in cultured excised tomato roots but in lower concentrations than in seedling radicles of the same age. The alkaloid was not detected in 'spent' root medium. Newly-initiated callus cultures of hypocotyl, radicle and cotyledon origin produced roots, and tomatine was isolated from both roots and callus. Roots contained more tomatine than callus, but neither contained as much as the organ explants from which the cultures were initiated. The number of roots produced decreased with time, as did also the tomatine content of the callus tissues. After 447 days, when no organized structures were produced by callus cultures, tomatine was not detected. An established hypocotyl callus contained small amounts of tomatine when grown on certain nutrient media, but a chlorophyllous sub-isolate of this callus did not produce detectable quantities of the alkaloid. Tomatine was not detected in an established root callus isolate or in suspension cultures initiated from established, tomatine-containing hypocotyl callus.     

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5?mg L^?1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200?mg L^?1 of chitosan was found to be 5.9?×?(6.5?mg g^?1 DW) higher over control cells. Treating the cells with 2?mg L^?1 of CCC, after 7?days, led to 149.3× enhancement of B content (19.4?mg g^?1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.     

The relation of biotic and abiotic interactions to eutrophication in Tjeukemeer,The Netherlands

During the summer months of 1974–1985 chlorophyll-a and total P concentration, biomass of Daphnia hyalina, smelt Osmerus eperlanus, bream Abramis brema and pikeperch Stizostedion lucioperca, water temperature and water intake from lake IJsselmeer were monitored in Tjeukemeer. During this period there were manipulations with the bream and pikeperch stocks as a consequence of the termination of a gill-net fishery in 1977, and larval smelt immigrated each year from the large lake IJsselmeer and contributed largely to the yearly smelt recruitment.The correlation matrix of the nine variables mentioned above showed a positive correlation between bream and chlorophyll-a, but surprisingly a negative one between smelt and chlorophyll-a. The latter can only be explained when smelt is the dependent variable. In a multi-linear regression there was a negative effect of temperature, chlorophyll a and pikeperch on smelt and a positive effect of water intake. Daphnia hyalina was negatively influenced by the biomass of smelt and the water intake of lake IJsselmeer. The positive relation of Daphnia hyalina and chlorophyll-a was probably related to better survival chances of D. hyalina in an Oscillatoria-rich environment when smelt is the most important predator. An increasing biomass of bream coincided with higher total-P levels and probably contributed to higher chlorophyll-a levels.     

A comparison of the sites of phytoalexin accumulation and of biosynthetic activity in potato tuber tissue inoculated with biotic elicitors

Aged discs cut from Kennebec potato tubers were inoculated with one of the following: an elicitor preparation from mycelia of Phytophthora infestans race 4, zoospores from either race 4 or race TY complex of this fungus, or sodium arachidonate. At 24 hr intervals after inoculation, four successive 0.5 mm thick layers of tissue were cut from the discs. This tissue was analysed for accumulated phytoalexins and also used to prepare cell-free enzyme systems for lubimin biosynthesis. In tissue treated with either the elicitor preparation or race 4 zoospores, levels of phytoalexin accumulation were highest in the first layer of tissue. Surprisingly, however, cell-free lubimin biosynthesis from [1-¹⁴C]isopentenyl pyrophosphate was also generally greater in preparations derived from the first 0.5 mm of tissue. Accumulation of phytoalexins in tissue inoculated with zoospores from race TY complex was very low, whereas cell-free biosynthetic activity was initially comparable to that seen in preparations from tissue treated with the elicitor preparation. By the end of the experimental period lower layers of tissue from discs treated with sodium arachidonate contained the highest levels of phytoalexins and yielded cell-free enzyme preparations with the greatest lubimin biosynthetic activity.     

Carbon dioxide and ethylene evolution in the culture atmosphere of Magnolia cultured in vitro

Subcultured explants of Magnolia soulangeana Soul, were incubated in tissue culture containers fitted with different types of closures. Type of closure affected the CO₂ concentration, with levels as high as 14% CO₂ being detected. The ethylene concentration increased gradually with time, to as much as 2–3 ppm after 9 weeks. There was a large variation in the composition of the atmosphere within the containers of any one type. The way by which a container was closed influenced exchange of the inner gas phase with the surrounding atmosphere and was important in determining the development of the cultured tissues.     

Rates of uptake and metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by cultured leaf segments at different stages of development in wheat

Immature leaf tissue of Triticum timopheevi Zukh. responded to supplied auxin and showed cell division in culture. The rates of uptake and of metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by such tissues were measured and compared with those of mature auxin-unresponsive tissue. The purpose of these experiments was to determine whether or not the concentration of auxin in cultured mature tissue was a factor limiting the cell division response to auxin. The data indicate that neither alterations in rates of uptake nor alterations in rates of metabolism could explain the loss of responsiveness to auxin which apparently occurs during cell differentiation. The results are discussed in the context of the view that changes in cell sensitivity to growth substances and not only the concentration of these compounds, play an important role in plant growth and development.     

Patterns of Fusarium community structure and abundance in relation to spatial, abiotic and biotic factors in soil

Members of the Fusarium genus are important components of many plant–soil systems worldwide and are responsible for many crop diseases. Knowledge of the relative influence of biotic and abiotic factors on this genus is therefore of broad economic and ecological importance. In order to address this issue, we examined Fusarium communities in soils nearby apparently healthy and symptomatic asparagus plants in 50 fields scattered in four agricultural regions of Québec, Canada. Fusarium community structure and abundance were assessed using genus-specific PCR-denaturing gradient gel electrophoresis and CFU counts, respectively. Multivariate statistical analyses were used to detect community patterns related to spatial, abiotic and biotic factors. Results suggested that Fusarium community structure (i.e. the presence and absence of the different Fusarium sequence variants in the samples) in soil is mainly related to biotic factors (arbuscular mycorrhizal fungi and bacterial community structure), whereas Fusarium abundance is more closely related to abiotic factors (mainly clay, organic matter, NH₄, Na and Cu). Some degree of influence of spatial patterns was also observed on both Fusarium community structure and abundance with, for instance, a large regional variation in Fusarium community structure. However, Fusarium community structure was not directly related to the disease status of nearby asparagus plants.     

High potential of adhesion to abiotic and biotic materials in fish aquaculture facility by Vibrio alginolyticus strains

Aims:  The ability of Vibrio alginolyticus strains isolated from Sparus aurata and Dicentrarchus labrax nursery to adhere to epithelial cell lines (Hep-2 and Caco-2), fish mucus and their ability to form a biofilm on different surfaces (glass, polystyrene, polyethylene and polyvinyl-chloride) was investigated in this study.
Methods and Results:  The extracellular products were rich in enzymes and the strains were haemolytic on Wagatsuma agar and possessed several hydrolytic exoenzymes such as proteases, DNase and lipases. Most strains tested were multiresistant to the 17 antibiotics tested including those used in the farm to treat vibriosis.
Conclusions:  These bacteria were able to form a biofilm on all the surfaces tested and the cell density was the highest on the PVC surface followed by that on the glass slides, polystyrene and the polyethylene surface. More than 50% of the tested strains were adhesive to the epithelial cell lines (Hep-2 and Caco-2).
Significance and Impact of the Study:  These properties allow these bacteria to survive, proliferate and persist in all stages of fish rearing nursery even after seawater treatment with UV light.     

Differences in the contributions of dietary water to the hydrogen stable isotope ratios of cultured Atlantic salmon and Arctic charr tissues

Hydrogen stable isotopes of animal tissues are well established tracers of migration ecology in terrestrial ecosystems. Recent research has highlighted δ²H as a potential tool in studies of aquatic ecosystems, particularly as a robust tracer for quantifying the importance of allochthonous subsidies. Although the use of δ²H has clear potential, some uncertainties remain, in particular with regard to the contribution of dietary water to consumer δ²H. Here, we quantify the contribution of dietary water to δ²H in two salmonid fishes, Atlantic salmon (Salmo salar L.) and Arctic charr (Salvelinus alpinus L.), reared on diets of known isotopic composition. Furthermore, we examined the capacity of fins (adipose and caudal) to provide a non-lethal means of estimating consumer δ²H. The proportion of deuterium derived from environmental water of all tissue was substantial in both Atlantic salmon (mean = 0.43 ± 0.1 SD) and Arctic charr (mean = 0.48 ± 0.15 SD) but varied considerably between both individuals and tissue type. White muscle proved to be the least variable of the tissues analysed. Although fins proved to be a possible non-destructive substitute, a degree of caution is recommended with their use, as the proportion of dietary water contributing to the deuterium of fins was considerable more variable.     

Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems

Optimization of in vitro culture system for the expansion and the maturation of male germ cells to post meiotic stages is a valuable tool for studies exploring spermatogenesis regulation and the management of male infertility. Several studies have reported promising results of mouse spermatogonial stem cells culture in three-dimensional (3D) culture systems and a subsequent production of sperm. In the present study, we investigated the capacity of a three-dimensional soft agar culture system (SACS) supplemented with Knockout Serum Replacement (KSR) in colony formation and inducing human germ cells to reach post-meiotic stages. Testicular cells from testes of brain -dead donors were first cultured for three weeks in proliferation medium. The cells were subsequently cultured in the upper layer of the SACS (3D group) in a medium supplemented with KSR and hormones, and the results were compared with that of a two-dimensional (2D) culture system. We found that the number and diameter of colonies and the levels of expression of Scp3 and Integrin α6 in the 3D culture group were significantly higher than in the 2D group. Our findings indicate that SACS can reconstruct a microenvironment capable of regulating both proliferation and differentiation of cell colonies.     

Effect of medium acidity on growth and rooting of different plant species growing in vitro

Micropropagated Choisya, Daphne, Delphinium, Hemerocallis, Hosta, Iris and Photinia were found to adjust the pH of Murashige and Skoog's plant tissue culture medium (initial pH 5.6 or 3.5) to different values depending on the species. When plant growth and rooting rates were determined after plants had been grown on media initially adjusted or buffered to values between 2.6 and 5.7 the different plant species were also found to have distinct pH requirements for optimal growth and/or rooting rates.Abbreviations MS Murashige & Skoog's (1962) medium - MS19 MS with additionally 10 g l^–1 sucrose - 80 mg l^–1 adenine sulphate and 130.9 mg l^–1 NaH₂PO₄ - BA 6-benzyladenine - NAA 1-naphthyl-acetic acid - IBA 3-indole-butyric acid - IAA 3-indole-acetic acid - 2iP N₆(2-isopentyl) adenine     

Factors affecting the initiation of mini-rhizomes from Trillium erectum and T. grandiflorum tissues in vitro

Leaf and stem explants of Trillium grandiflorum and T. erectum produced mini-rhizomes (MRs) in vitro which gave rise to shoots and roots. The apical portion of the stem and the basal portion of the leaves were the most effective explants from these tissues, while stem tissue was more responsive than leaf tissue. The best response with both species was observed on half-strength MS basal medium supplemented with cytokinin and auxin. T. erectum was more responsive than T. grandiflorum overall, and in some cases produced MRs in the absence of growth regulators. Culture at 21°C appeared to stimulate growth from T. grandiflorum tissues, compared with controls at 27°C, whereas the outgrowth of shoots from MRs was inhibited in both species at 21°C. In vitro production of MRs could provide a more rapid, alternative propagation method for these species than traditional methods.     

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, α-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth’s MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300 μg per ml), hydrocortisone (1 μg per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeably promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporation of collagen-promoting factors to the in vitro environment particularly with regard to the controlling role implicated for collagen in a variety of biological processes. Some of the work reported here was undertaken while A. H. Melcher was a member of the Department of Dental Science, Royal College of Surgeons of England, London, England.     

Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata

Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO₃) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.