Chris H. Bornman

Avoidance of precipitation and carbohydrate breakdown in autoclaved plant tissue culture media

The extent of breakdown of fructose and glucose derived from sucrose in the medium of Murashige and Skoog (1962) during autoclaving was investigated by polarographic measurement. Although not present in the original MS medium but often used in place of FeSO4 + Na2-EDTA, FeNa-EDTA was found to be primarily responsible for catalyzing the breakdown of these monosaccharides. It would therefore be good practice to autoclave FeNa-EDTA separate from the carbohydrate constituents of the medium in order to reduce the formation of toxic substances derived from the latters breakdown. Autoclaving FeNa-EDTA separately has the additional advantage of preventing precipitation of certain micronutrient elements. Further precipitation can be avoided by autoclaving FeNa-EDTA and KH2PO4 together, but separately, from other components of the medium. By eliminating precipitation and minimizing the breakdown of monosaccharides during autoclaving, it is possible to improve the quality of the medium without resorting to sterilization by filtering.

Organogenesis in Cultured Pinus sylvestris Tissue

Summary Coumarin (10 µM), alone or in combination with IAA and BAP, induced a relatively high rooting-frequency in plumule explants of 14-day-old Pinus sylvestris seedlings; its effect on needle explants was less marked and erratic. Adventitious buds were readily induced in needle explants that consisted of the entire short shoot or dwarf spur, the retention of the growth-arrested shoot apex seemingly having an enhancing effect. Needle explants without the dwarf spur produced prolific callus but were very much less responsive organogenically.

Effects of synthetic cytokinins on levels of endogenous cytokinins and respiration patterns ofBeta vulgaris cells in suspension

Respiration patterns and growth of cytokinin-dependent cell suspensions ofBeta vulgaris L., precultured in media with or without three different synthetic cytokinins [benzyladenine (BA), kinetin (KIN), and thidiazuron (TDA)], were compared. The content of endogenous cytokinins, especially zeatin and isopentenyladenine, as well as the dry mass yield, were dependent on the kind of synthetic cytokinin present in the culture medium and decreased in the following order: thidiazuron, kinetin, benzyladenine, no cytokinin. The apparent capacity of the alternative pathway, as measured after blocking of the cytochrome pathway by cyanide, was inversely proportional to the content of endogenous cytokinins. Some synthetic cytokinins (e.g., benzyladenine), when exogenously applied, are known to inhibit selectively the alternative pathway. However, this does not necessarily imply that the mechanism of action of endogenous cytokinins on the respiration pattern is limited to a single effect on the alternative pathway. Multiple effects on oxidative processes cannot be excluded.

Effects of ultraviolet-radiation on viability of isolated beta-vulgaris and hordeum-vulgare protoplasts

Summary Estimates of viability as measured by vital staining with fluorescein diacetate were carried out on freshly isolated and partially aged (16-hour-old) Beta vulgaris and Hordeum vulgare mesophyll protoplasts following irradiation with UV-B. Damage to the photosynthetic system by UV-B was determined by delayed light emission (DLE). In the case of freshly isolated protoplasts Beta was approximately 30% more susceptible than Hordeum following 3h irradiation, with viability decreasing from 90% to 40%. After storage of protoplasts on ice for 16 h UV-B radiation markedly depressed viability in both species, but in the case of Hordeum there was a substantial initial loss of nearly 70% in viability over the first hour of irradiation. The first 10 min of UV-B radiation decreased the intensity of DLE by 40% without appreciably affecting the decay rate. Longer treatment times did not give a proportional effect so that even after 60 min of UV-B the inhibition did not exceed 60%. This suggested that although the enzyme system responsible for FDA hydrolysis may be partially inactivated (viability was 75–80% as compared with 90% in the control), the UV-B did not penetrate the innermost parts of the chloroplasts, but left some thylakoids undamaged.

Cyanide-initiated oxygen consumption in autoclaved culture medium containing sugars.

The consumption of oxygen initiated by KCN in an autoclaved sugar-containing rinse medium with protoplasts is described. The effect of autoclaving on several sugars was examined. Fructose solutions, followed in decreasing order by glucose, sucrose and sorbitol, were found to contain the largest amount of degraded products that could react with oxygen in the presence of KCN. Mannitol was found to be stable under the autoclaving conditions used in this investigation. KCN generally has an inhibitory effect on respiration, but in some plant tissues, respiration is stimulated by it. Under certain circumstances the degradation artefact described here may confuse interpretation of the results of respiration measurements. The use of autoclaved media containing sugars should be avoided in respiration studies that involve the application of KCN.

Immobilization of protoplasts by anchoring to microcarriers : Chris H. bornman and anders zachrisson.

Datura innoxia cell suspension-derived protoplasts were anchored to Cytodex 1 microcarriers pre-swollen in buffered concanavalin A. As many as 34 protoplasts were estimated to attach per microcarrier, in comparison to a potential 47 as determined from a model based on random anchorage. Fluorescein diacetate was used as localizing agent as well as to assess viability. When included in the swelling medium fluorescence was observed almost instantaneously, first in the protoplast at its interface with the microcarrier, and later throughout the cytoplasm. However, the dye was not conjugated with the lectin, and leakage eventually resulted in fluorescence also of non-anchored protoplasts. Fluorescein-labelled concanavalin A on the other hand permitted detection of microcarriers but not of anchored protoplasts, suggesting the use of differentially fluorescing microcarriers, as an aid in identification of fusion partners.

Quality indicators for woodwind reed material

For the generation of sound, some woodwind musical instruments, e.g. oboe, bassoon, clarinet and saxophone, are provided with mouthpieces made from reeds. These reeds are the culms of Arundo donax, a tall, cane-like perennial grass. A general problem is that the material is of varying quality, yet externally differences cannot be observed. Hence, large proportions of the prepared reeds are unusable. One hypothesis is that the changes in quality are correlated with differences in the chemical and anatomical structure of the tissue. Therefore, a comparison of superior and inferior mouthpieces, used by professional musicians, was undertaken to determinate potential indicators of quality. Nuclear microprobe analysis of reeds was carried out and complemented by scanning electron and light microscopy. The elemental levels of Si, P, S, Cl, K and Ca were compared between good and poor mouthpieces using appropriate statistical tests. No statistically significant differences could be identified. Microscopical observations showed that partial occlusion of vessels by tylose formation was associated with material deemed unusable.

Fluorogenic compound hydrolysis as a measure of toxicity-induced cytoplasmic viscosity and pH changes

If a fluorogenic compound, such as fluorescein diacetate, is added to a water solution containing living cells it becomes hydrolyzed by intracellular esterases into a fluorochrome whose fluorescence can be used to monitor the cytoplasmic pH and the cytoplasmic viscosity of the cells. In this paper we have used this technique to measure the effects of different concentrations of Co2+ and Cd2+ ions on the cytoplasmic pH and the cytoplasmic viscosity of a single cell culture. Our results indicate that the observed decrease in the efficiency of the intracellular hydrolyzation of fluorogenic substances in the presence of different concentrations of heavy metals could be caused by both a decrease in the cytoplasmic pH and an increase in the cytoplasmic viscosity. A decrease in cytoplasmic pH would decrease the effectiveness of the intracellular enzymes, whereas an increase in cytoplasmic viscosity would decrease diffusion which would also reduce the effectiveness of the reaction. The dependence of the reciprocal of the cytoplasmic viscosity on the concentration of these metals correlates well with published results on their toxicity.

Welwitschia mirabilis: Fine Structure of the Germinating Seed V. The Quiescent and Imbibed Gametophyte Interface

Summary Gametophyte cells in the gametophyte-embryo interface zone contain less densely packed reserves and smaller protein bodies (mean diameter 1.2 ,µm) as compared with contiguous and deeper-lying cells. They have more residual cytoplasm, numerous amyloplasts, mitochondria, few microbodies, fragments of ER, but no dictyosomes. Upon imbibition the protein bodies degrade rapidly, starch reserves decline and disappear within 24 h, microbodies increase in number and the ER develops longer single profiles. After 3 days protein bodies start to fuse, microbodies multiply, the ER becomes extensive and sheetlike, and dictyosomes are observed. Between days 4 and 6 ER-derived minivacuoles are autophagically active, accumulating membrane-bound fibrillar material and engulfing lipid bodies; the cell walls have thickened; and mitochondria exhibit a close spatial relationship with lipid bodies. The outer cells of the mature feeder are crushed against the gametophyte. With imbibition physical wetting of outer gametophyte cells precedes that of interface cells, yet nuclear activation and subsequent cellular activity is accelerated in the latter and stand in sharp contrast to the slower metabolism of the former.

Chloroplast ultrastructure and fluorescence response of oilseed rape containing male-sterile radish cytoplasm

SummaryThe production of hybrid seed is facilitated if one parent possesses a male-sterile cytoplasm. Introduction of the cytoplasm of male-sterile radish (Raphanus sativus L.) into rapeseed (Brassica napus L.) results not only in transfer of the desirable male-sterile trait but induces a chlorophyll defect in the backcrossed male-sterile plants. In this study we show that the defect manifests itself in two different ways in the alloplasmic plants: a) smaller and fewer chloroplasts with an impaired ultrastructure and b) an increase in chlorophyll fluorescence. Defective chloroplasts were characterized by a reduction in both the number and size of grana, the latter due to poor stacking of thylakoids and with frequent discontinuity in the intergranal thylakoid systems. The changed chloroplast morphology and the increase in chlorophyll fluorescence are probably the cause of the lowered photosynthetic efficiency associated with the alloplasmic plants. We propose that the deficiency is the result of incompatibility between the genomes of the radish chloroplast and the rapeseed nucleus. Supporting this hypothesis are studies of male-sterile rapeseed plants in which, by protoplast fusion, the radish chloroplasts were substituted by those of normal male-fertile rapeseed. Such plants showed complete restoration of their photosynthetic potential and displayed both normal chloroplast ultrastructure and normal levels of chlorophyll fluorescence.