Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

Summary Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20–30 days. Cells were cultured in either -minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 g/ml ascorbic acid, or the above medium supplemented with either 10 mM Na--glycerophosphate, 10^-8 M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Glaprotein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both -glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Glaprotein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both -glycerophosphate and, particularly, dexamethasone.     

Occurrence of rye (Secale cereale) 350-family DNA sequences inAgropyron and otherTriticeae

Evidence is presented that in the R and P genomes (Secale cereale andAgropyron cristatum, respectively) of theTriticeae there exist closely related 350-family DNA sequences in the terminal heterochromatin. This observation is compared to the relationships between these two genomes derived from a comparison of theNor and5 S DNA loci as well as the available data on morphological characters, chromosome pairing, and isozyme studies. It is concluded that the R and P genomes are not closely related and that the common presence of very similar 350-family DNA sequences reflects the parallel amplification of this family of DNA sequences.     

Chromium in plants

Chromium is an essential trace element and is associated with some biological pathways, especially with glucose tolerance. For these reasons, we decided to determine the concentration of chromium in two sets of Brazilian medicinal plants. The first group consisted of plants that are considered as antidiabetic, whereas the second included plants that do not have this therapeutic property. The concentration of chromium was determined by flameless atomic absorption. All the plants analyzed contain chromium in the normal range for this element, but the hypoglycemic plants contain more chromium than the others (1–4 μg/g compared to 0.5–1.5 μg/g).     

Effects of traces of rare earth elements on the protozoanBlepherisma and on mice

The growth of the protozoanBlepherisma is stimulated by Lanthanum (La) at concentrations as low as 0.32 ppm. In mice Yttrium (Y) and Ytterbium (Yb) are absorbed, accumulated, and metabolized. Both rare earth elements (RE) exhibit a high affinity for teeth and bones, accumulation occurs and metabolism is slow. In the livers of RE-exposed mice, concentrations are variable. The liver is apparently capable of absorbing and discharging RE in a manner depending on metabolic activity. The main route of discharge for ingested REs is the alimentary canal. Exposure of pregnant mice to RE leads to rapid placental transfer of RE; 14.1% of the total amount of RE administered was detected in newborn mice. Young, developing organisms appear to be especially susceptible to RE accumulation.     

In vitro propagation and subsequent nodulation of the actinorhizal Hippophae rhamnoides L.

Micropropagation of the actinorhizal plant Hippophae rhamnoides L. (sea-buckthorn) was achieved on Murashige and Skoog (MS) medium supplemented with 1 M of benzylaminopurine (BA). A multiplication frequency of three to five shoots per explant was observed after 28 days. Rooting of these shoots was achieved in a medium containing 1/4 strength MS without growth regulators. The rooted plants were transferred to Turface R artificial substrate and inoculated with pure cultures of two Frankia strains. These plantlets subsequently developed nodules which fixed nitrogen.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

Histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets

Calli have been initiated from young leaves of in vitro grown sugarcane shoots. Histological examination has shown that the two types of calli induced (nodular and friable) originated from different regions of the explants and were cytologically different.This study has shown an obvious relation between the developmental stage of the excised tissue and the potential of plant regeneration of the in vitro initiated callus culture. Nodular calli were obtained from bases of the fast-growing young leaves while their more mature parts of the older leaves only produced friable calli. High-frequency plant regeneration via somatic embryogenesis was obtained from nodular calli while friable calli rarely produced plantlets.     

Effects of auxins and cytokinins on epigenetic instability of callus-propagated Kalanchoe blossfeldiana Poelln.

A main problem in the vegetative propagation of ornamental plants in vitro is the epigenetic instability of cells removed from their organized environment. With calluses of leaf explants of Kalanchoe blossfeldiana Poelln., cv. Yucatan, the role of plant growth regulators (PGRs) in the occurrence of fasciation was studied.In various combinations of auxins and cytokinins, the auxin 2,4-D (2,4-dichlorophenoxyacetic acid) gave only deformed, inseparable shoot primordia. The most rapid callus induction with regeneration of well-developed sprouts was obtained with the natural IAA (indoleacetic acid) and Z (zeatin).As a first symptom of fasciation, aberration in decussate phyllotaxis can be observed. At increasing concentrations of IAA + Z, this symptom gradually decreased but fasciation proper increased. The optimum concentration was at 1 M for both PGRs. Reduction of exposure to the PGRs from six to three weeks reduced the epigenetic instability.     

In vitro tuberization in white yam (Dioscorea rotundata Poir)

Nodal cuttings of white yam were induced to produce microtubers on a MS-revised medium supplemented with various concentrations of sucrose, 20 mgl^–1 L-cysteine, 0.5 mgl^–1 kinetin and 0.7% agar. The frequency of tuberization was affected by the daylength, which is optimal at 12 and 16 h of light depending on the sucrose concentration. The microtubers were planted in a seed bed and grown to maturity. The importance of in vitro tuberization of yam as a means of international germplasm distribution or exchange as well as for the propagation of planting material is discussed.