Protoplast-derived streptomycin resistant plants of the forage legume Onobrychis viciifolia Scop. (sainfoin)

Approximately 10⁶ protoplast-derived cell colonies of sainfoin were stressed with streptomycin and two resistant colonies were recovered. Plants regenerated from these colonies could be recallused on streptomycin-containing medium three years after growth in the absence of the antibiotic.Ultrastructural studies showed cells of resistant callus grown in the presence of streptomycin to contain chloroplasts with internal thykaloids and grana. Such mutant plants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.Abbreviations BAP 6 - benzylamino purine - NAA - napthaleneacetic acid     

The influence of sucrose, 2,4-D,and kinetin on the growth,fine structure,and lignin content of cultured sycamore cells

Summary When suspensions of sycamore cells are cultured in a synthetic medium containing 1.0 mg/l 2,4-D and 0.25 mg/l kinetin, maximum cell yield is obtained with an initial concentration of 6 per cent sucrose. There is a progressive increase in dry weight per cell, decline in extractive-free weight as a percentage of cell dry weight and increase in lignin content per cell as the initial sucrose concentration is increased from 1 per cent to 15 per cent. The percentage of lignin in the extractive-free cell residue is further enhanced by increasing the level of 2,4-D to 10 mg/l or by growing the cells in an auxin-free medium containing 10 mg/l kinetin.When the cell suspensions are treated with phloroglucinol/HCl it is found that only a proportion of the cells contain lignin, that this lignin occurs in the protoplasts and in plates between the cells, and that lignin is present in the culture medium.Electron micrographs confirmed the absence of any secondary wall such as is characteristic of tracheary elements. Cells cultured in the presence of 6 per cent sucrose or higher levels showed numerous amyloplasts and frequently the presence of electron opaque material. This occurs in the irregular but not frequent wall thickenings, as droplets in the vacuoles and as amorphous sheets between the cells. Pictures showing such electron opaque droplets clustered on the inner face of the tonoplasts suggest that this material is formed in the cytoplasm and released into the vacuoles. In addition these cells are characterised by the presence of fine electron opaque granular material in their vacuoles and external to their protoplasts. Cultures richest in lignin showed the highest content of electron opaque globules in, and amorphous sheets between, the cells and it is suggested that these correspond to lignin or a lignin-hemicellulose complex. In the presence of 15 per cent sucrose many cells showing breakdown of organised structure were observed; they were characterised by the persistence of mitochondria and particularly of the amyloplasts and by their high content of the electron opaque material equated with lignin. This material was also present in the dead cells.     

Molting in a parasitic nematode, Phocanema decipiens—VII. The mode of action of the ecdysial hormone

The cycle of aminopeptidase activity demonstrated by histochemical methods in the activated excretory gland could not be detected in homogenates. In the electron microscope, the secretory granules in dormant glands were dense and irregular in shape and the mitochondria elongate, relatively dense, and with a crenellated outer membrane. The excretory gland was activated when the neuroendocrine system was stimulated by farnesyl methyl ether. In activated glands the secretory granules became larger, less dense and the membranes began to fuse with membranes of the ductules which ramify through the gland. The mitochondria became swollen. Aminopeptidase activity was displayed by a large uniformly less dense granules but not by the denser granules, and was seen in the endoplasmic reticulum, Golgi apparatus and in the lumen of the ducts. It is suggested that the ecdysial hormone from the neurosecretory cells sets in train a sequence of events which leads to entry of water into the gland and consequent activation of the enzyme in the granules, and to changes in the membranes of the granules which facilitate fusion with the membrane lining the ducts.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Plant regeneration from seedling explants and cotyledon protoplasts ofGlycine argyrea tind

Summary Plants have been regenerated from nodular, green callus derived from cotyledon, petiole and leaf lamina explants ofG. argyrea, a perennial relative of the soybean (G. max). The degree of response obtained was governed primarily by the genotype used, accession G1626 proving the most responsive. Shoots were also recovered from about 6.0% of cotyledon protoplasts of this genotype. The implications of these results are discussed in relation to genetic manipulations using this species.     

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl^–1 BAP, 0.05 mgl^–1 IBA and 50 gml^–1 kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IBA indole-butyric acid - PAGE polyacrylamide gel electrophoresis - NPTII neomycin phosphotransferase II - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecylsulphate