Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara)

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA3 gibberellic acid - NAA -naphthaleneacetic acid - PAS periodic acid Schiff - PCM protoplast culture medium - PCV packed cell volume     

Adherence to epithelial cells and ultrastructure of fosfomycin-resistant mutants of group A streptococci

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.     

The adhesin structures involved in the adherence of group B streptococci to human vaginal cells

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.     

A Comparison of Internal Standards for Plant Cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

Combination of lamin immunocytochemistry and in situ hybridization for the analysis of chromosome copy numbers in tumor cell areas with high nuclear density

We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections.     

Purification and characterization of Kurloff cell sialoglycoproteins with acid phosphatase activity

The majora2–6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange andSambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1)S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the majora2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4–3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylatedN-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but alsoa2-6-linked sialic acid residues themselves may participate in natural killer activity.