Lipids in plant tissue cultures IV. The characteristic patterns of lipid classes in callus cultures and suspension cultures

Lipids from callus cultures and suspension cultures of higher plants constitute 5 to 8% of the dry tissue's weight.The predominant lipid classes are the sterols, steryl esters, steryl glycosides and esterified steryl glycosides. Considerable amounts of a variety of sterylglycolipids, whose structures are not completely elucidated, are also present. Triglycerides and phospholipids occur in small proportions, whereas monogalactosyl diglycerides, digalactosyl diglycerides and sulfoquinovosyl diglycerides are present only in traces, if at all.β-Sitosterol is the predominant constituent sterol, stigmasterol and campesterol as well as a variety of as yet unidentified sterols occur in smaller proportions. The major constituent fatty acids are palmitic, oleic, linoleic and linolenic acids. Saturated very long-chain fatty acids are found in smaller proportions. Unusual fatty acids, such as epoxy acids, which occur in the seed lipids of certain plants, are not found in tissue cultures derived from these plants. Clucose and traces of galactose are the only sugars obtained by acid hydrolysis of the glycolipids occurring in plant tissue cultures.     

Lipids in plant tissue cultures. III. Very long-chain fatty acids in the lipids of callus cultures and suspension cultures

The lipids in plant tissue cultures contain in addition to the common saturated and unsaturated fatty acids even- and odd-numbered fatty acids having chain-lengths up to 26 carbon atoms.     

Cytological and ultrastructural changes induced in anther and isolated-microspore cultures in barley: Fe deposits in isolated-microspore cultures

To gain further insight into the role played by sporophytic anther tissues in the early stages of the androgenic process, we have compared the cytology and ultrastructure of barley embryogenic pollen grains obtained by anther culture with those obtained by isolated-microspore culture. The microspores behaved similarly in both culture systems but ultrastructural studies detected a significant difference: the presence of electron-dense deposits on the intine of embryogenic pollen grains generated by isolated-microspore culture compared to their absence in grains generated by anther culture. To discover the nature of these deposits, we applied proteinase K and EDTA treatments to ultrathin sections. We also subjected the deposits to X-ray microanalysis and found that they contained iron. Anthers and isolated microspores were cultured in media containing different concentrations of iron so as to evaluate the presence of these deposits on the intine. Deposits were not found in anther cultures at any iron concentration used or in microspore cultures when concentrations were lower than 40 mg/L. The Fe deposits on the intine appear to derive from an excess of Fe in the isolated-microspore culture medium which, if allowed to pass through the cell wall, could well be toxic to the embryogenic development of the microspores.     

Thiomolybdates in rumen contents and rumen cultures

Examination of direct and (Cu)-difference spectra of i) the aqueous supernatants of in vitro cultures of bovine rumen contents incubated with MoO42- and potential sources of S2- and ii) samples drawn directly from the rumen of animals receiving high Mo diets yielded evidence of the presence of thiomolybdates. Only MoS42- was detected in the soluble phase of in vitro cultures. Although intense and variable background absorbance precluded full characterization of thiomolybdate species in samples drawn directly from the rumen, both spectral data and the biochemical and clinical responses of animals given high Mo diets were consistent with the conclusion that MoS42- rather than MoOS32- was the predominant thiomolybdate species present in the aqueous phase. Addition of Ca2+ either to rumen cultures before incubation or as a supplement to diets high in MoO42- content inhibited the appearance of MoS42- in the aqueous phase. Evidence of the sequestration of MoS42- and MoOS32- by particulate or microbial fractions of rumen contents is considered in relation to the inhibitory action of Mo upon Cu absorption by ruminants.     

Convection and diffusion in tissues and tissue cultures

The relative importance of transport by diffusion and convection in permeable tissues is investigated in three-dimensional structures. The transport of a solute takes place from a number of sources embedded in the tissue, to a number of sinks similarly embedded. An enhancement factor E is defined to be the ratio of the transport rate in the presence of a pressure difference between the sources and sinks, to the transport rate without a pressure difference. It is shown that E is a unique function of a parameter W, which characterizes the properties of the tissue and the pressure difference. This relation is independent of the number or the geometries of the sources and sinks.This relation is compared with experimental data obtained in a hollow fiber tissue culture device, with two sets of hollow fibers kept at different pressures. This relation is also used to estimate the importance of convection in vivo for a wide range of mammalian tissues and solute molecules.     

Hemopoiesis in spleen and bone marrow cultures

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics in vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.     

Neuronal and glial differentiation in reaggregation cultures

Summary Dissociation and reaggregation cultures from different portions of the chick embryo neural tube were made, and the resulting aggregates were fixed for electron microscopy after 1, 5, 8, 14, 16 and 22 days in vitro. All cultures (pure aggregates of telencephalon, optic lobe or neural retina, and combined aggregates made from mixtures of optic lobe plus neural retina or optic lobe plus telencephalon) show a common timing of neuronal and glial morphological differentiation.During the first week in vitro, some cells developed neuronal characteristics in the absence of morphological evidence of glial differentiation. Numerous axonic processes usually formed fascicles with all the fibers running parallel to each other. Axonic growth cones were abundant and a few immature synapses were also present. The second week in culture was characterized by the disappearance of growth cones and the increase in number and morphological maturation of synapses. Morphologically detectable glial differentiation began by the end of this week, and during the third week almost every neuronal element, including the axonic fascicles, became associated with glial cells showing astrocytic features.The authors are indebted to Mrs. N.A. de Pinto for her competent technical assistanceA.M.S. and R.A. are members of the Carrera del Investigador del Consejo Nacional de Investigaciones Cientifícas y Técnicas de la República ArgentinaThis work was supported by a grant from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, and by NIH grant 5 ROI NS 06953-09 NEUA to Prof. Dr. E. De Robertis     

Ultrastructural study of Pneumocystis carinii in explant cultures of rabbit lung and in cultures with and without feeder cells

Pneumocystis carinii-parasitized lung explants were obtained from corticoid-treated rabbits and maintained in vitro. Twenty-one days after the beginning of explant cultures, the ultrastructural morphology of trophozoite, precyst and cyst forms was normal as compared to the in vivo ultrastructure of P. carinii from infected rabbit. However, after the 36th day, only altered forms of P. carinii were observed. Lung tissue showed only minor alterations. Intracytoplasmic lamellar inclusions were observed in type 2 alveolar cells from which they were released. While the total number of parasites increased approximately 4-fold from day 0 to day 41, trophozoite counts increased approximately 6 times. Pneumocystis cells from inocula and supernates of cultures with and without Vero cells showed important ultrastructural alterations.     

Lipids in plant tissue cultures. VII: Heterotrophic and mixotrophic cell cultures of Chenopodium rubrum

Mixotrophic cell cultures of Chenopodium rubrum were found to synthesize 5 to 33 times more monogalactosyldiacylglycerols and 5 to 16 times more digalactosyldiacylglycerols than heterotrophic ones. The monogalactosyldiacylglycerols and digalactosyldiacylglycerols from mixotrophic cultures contained higher levels of linolenic acid as compared to heterotrophic cultures. It is concluded that the active synthesis of these galactolipids with high levels of constituent linolenic acid is associated with the onset of photosynthesis in plant cell cultures, as is the case in intact plants.     

Callus cultures for phytometabolism studies: phytometabolites of 3-trifluoromethylphenol in Lemnaceae plants and callus cultures

Plant callus cultures have the potential to advance phytoremediation science by allowing study of cellular phytometabolism in absence of sorption, translocation, microbial degradation, and other phytoremediation processes; however, studies demonstrating the applicability of results from callus cultures to whole plants are limited. The aim of this study was to evaluate the feasability and applicability of using callus cultures to study phytometabolism. This aim was accomplished through evaluation of induction and growth of Lemnaceae callus cultures and comparison of phytometabolism in callus cultures and whole plants. Four out of eight published methods for callus culture of Lemnaceae successfully induced callus cultures that exhibited doubling times of 1.7 to 23 wks. Callus cultures and whole plants of Landoltia punctata and Lemna minor metabolized 3-trifluoromethylphenol (3-TFMP) through conjugation with glucopyranoside, malonyl-glucopyranoside, and glucopyranosyl-apiofuranoside. However, concentrations of metabolites were approximately 10 times less in callus cultures than in plants. While results demonstrated applicability of callus cultures results to whole plants, the low success rate of callus induction procedures, length of time required to produce substantial callus mass, and the low accumulation of metabolites in callus cultures may limit the feasibility of callus cultures for assessing phytometabolism.