Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell–cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions.     

T-DNA-insert-independent mutations induced in transformed plant cells duringAgrobacterium co-cultivation

Transformation frequencies were determined for 1n, 2n, and 4n Nicotiana plumbaginifolia protoplast cultures inAgrobacterium-mediated gene transfer experiments. An unexpected large drop (50%) in plating efficiencies was observed in the non-selected (control) 1n populations after transformation treatment with virulent strains. This effect was not observed in the 2n or 4n cultures or in the 1n cultures when treated with avirulent bacteria. The mortality was disproportionally high and could not be explained by the low (0.1–0.5%) transformation efficiency in the 1n population, indicating mutagenesis of the cell populations independently from the T-DNA insertions. Mutagenesis was also indicated in gene tagging experiments where nitrate reductase-deficient (NR^–) mutants were selected from haploidNicotiana plumbaginifolia protoplasts, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gene (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt), afterAgrobacterium co-cultivation. The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only after NR-deficiency had been established. Thirty-nine independent NR-deficient mutants were analysed further by Southern blot hybridization. There was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequently in cell populations which became transformed during the treatment than in the populations which did not. These observations suggest that transformation-competent cells undergo mutagenesis during theAgrobacterium gene transfer process not only as a result of stable integration events, but also through accompanying events that do not result in major changes in the mutated loci. The nature of these changes at the molecular level remains to be elucidated.     

Role of T Cells in the Mitogen-Induced Proliferation and Polyclonal Antibody Response of Murine B Cells

The effect of thymus-derived lymphocytes (T cells) on the responsiveness of bone marrow-derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in a nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti-sheep erythrocyte (SRBC) IgM hemolysin plaque-forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti-SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC. Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti-SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti-SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.     

Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium

Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding density of 17 cells/cm² with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.     

Fertile plant regeneration from protoplasts of meadow fescue (Festuca pratensis Huds.)

Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions.Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10^-3 to 10^-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.     

Antigen-Specific Proliferative Response of Peritoneal Exudate Lymphocytes Primed with Antigen and Bacterial Lipopolysaccharide: The Roles of Ia+ Accessory Cells and IL-2

In vitro antigen-specific proliferation was investigated in a lymphocyte population that had been taken from the peritoneal exudate cells (PEC) of C3H/HeN mice (Ia^k) primed in vivo with both bacterial lipopolysaccharide (LPS) and horse red blood cells (HRBC) and had been purified by passage through a nylon fiber column (Nfc). The proliferative response of the Nfc-passed lymphocytes primed with HRBC and LPS [T(HRBC + LPS) cells] depended on the dose of antigen in the cultures, and the response was higher than that of cells prepared from mice primed with HRBC alone [T(HRBC) cells]. No response was seen in the cells prepared from the LPS-primed mice [T(LPS) cells] or normal mice [T(N) cells]. The response of the T(HRBC) cells was abolished by previous treatment of the cells with anti-Ia^k antibody and complement (C), whereas the response of the T(HRBC + LPS) cells was retained after the same treatment, indicating that the Ia^– T(HRBC + LPS) cells can proliferate in response to antigen in spite of Ia+ accessory cell-depletion. Supernatants from the cultures of Ia^– T(HRBC + LPS) cells in the presence of HRBC showed abundant IL-2 activity, while those of Ia^– T(HRBC) cells did not. The IL-2 should be produced by the L3T4 cell population in T(HRBC + LPS) cells in response to antigen, since the previous treatment of the cells with anti-L3T4 antibody and C abrogated the production. On the other hand, the Ia^– T(HRBC + LPS) cells as well as the Ia^– T(LPS) cells could respond to IL-2 dose-dependently when recombinant IL-2 was added into the cultures, but the response of Ia^– T(HRBC) cells to IL-2 was very weak. The cell population responding to IL-2 in the T(HRBC + LPS) cells as well as T(LPS) cells must be AsGM1-positive or natural killer (NK) cells, since previous treatment of the cells with anti-AsGM1 antibody and C abrogates the response. Together these results suggest that L3T4 lymphocytes capable of producing IL-2 in response to HRBC antigen without Ia⁺ accessory cells are generated in the PEC of the mice after priming with LPS and antigen together, and the IL-2 produced by the L3T4 lymphocytes induces the proliferation of the LPS-primed AsGM1+ cells.     

Efficient plant regeneration from rice protoplasts in general medium

Summary We established an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant regeneration frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.Abbreviations AA Amino Acid medium (Muller and Grafe 1978) - MS Murashige and Skoog (1962) medium     

Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor-β1 with time in culture

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-β1 (TGF-β1) when treated within 1–2 days after plating. The purpose of the present studies was to examine the effects of TGFβ1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGFβ1 decreased dramatically as the cultures aged. The IC₅₀ for inhibition of colony forming efficiency was 0.18 pM when TGFβ1 was added 24 h after cell plating. When TGFβ1 treatment was begun on day 5 of culture, the IC₅₀ was 3–4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGFβ1 was begun on day 19, the IC₅₀ was 65 pM or > 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGFβ1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGFβ1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGFβ1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGFβ1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGFβ1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-α, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGFβ1 by about twofold at both early and late times in culture. This indicates that the changes in TGFβ1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events. © 1992 Wiley-Liss, Inc.     

Studies on the effect of protoplast density and genotype mixing on cell regeneration

Genotypes of Solanum tuberosum, assessed for their differential response to a protoplast regeneration protocol, were identified. This enabled comparisons to be made in relation to their abilities to regenerate into dividing cells and colonies. Protoplasts from genotypes with contrasting regeneration responses were plated as single or mixed genotype cultures at various plating densities in replicated, randomised experiments, and the effect on the regeneration of particular cell types observed. Protoplasts from single genotype cultures showed intergenotypic variation in the extent of cell regeneration and there were significant effects of genotype mixing and density × genotype mixing interactions. Overall, the effect of mixing was beneficial to a regenerating culture but significant density genotype mixing interactions showed only a positive benefit at the lowest plating density. The protoplast mixing phenomenon was not correlated with the behaviour of the same genotypes at the plantlet level in experiments with in vitro meristem cultures.     

Mouse epidermal cell cultures : II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10⁶ viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%.The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.     

Factors affecting transformation of cell cultures from three dicotyledonous pigment-producing species using microprojectile bombardment

Gene transfer methods were established for cell suspension cultures of sweet potato (Ipomoea batatas), ohelo (Vaccinium pahalae) and carrot (Daucus carota, two lines) using micro-projectile bombardment. Several parameters were studied (particle size/type, helium pressure, stage height, DNA concentration, pre-culture period) to determine which significantly affected transformation efficiency. All the physical parameters influenced transient gene expression, with particle size and type having the greatest effect. Cell culture age also affected transformation efficiency in all cell lines. Nuclear DNA conformation (relaxation) as measured by flow cytometry showed no change associated with culture age or transformation efficiency. Agrobacterium-based methods were also tested and in most experiments produced no GUS-expressing loci. Microprojectile bombardment will now be used to study anthocyanin biosynthesis in cell cultures as an alternative source of natural food colourants.     

Conditions for a high plating efficiency of free cell suspensions of Haplopappus gracilis (Nutt) gray

Summary Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 m. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency.     

Differential chromosomal fluorescence with 33258 Hoechst

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10⁶ viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%.The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

An improved procedure for plant regeneration from indica and japonica rice protoplasts

Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R₂ medium modified by the addition of 560 mg l^–1 of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l^–1) and -naphthaleneacetic acid (0.5 mg 1^–1), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GPFs growth promoting factors - NAA -naphthaleneacetic acid On leave from Department of Genetics, Haryana Agricultural University, Hisar, IndiaOn leave from Biotechnology Centre, Punjab Agricultural University, Ludhiana, India