In vitro culture of embryos of Cucumis spp.: heart-stage embryos have a higher ability of direct plant formation than advanced-stage embryos

Summary The ability of embryos at different developmental stages to form plants in vitro has been studied in cultivated Cucumis sativus L. and in the wild species C. zeyheri 2 x Sond. and C. metuliferus Naud. On MS medium containing 3.5% sucrose, 0.1 mg 1^–1 kinetin (Kn) and 0.01 mg 1^–1 indoleacetic acid (IAA), proembryos (0.03–0.05 mm) and early globular embryos (0.05–0.08 mm) from the wild species developed into plants in low frequencies of 8% and 21%, respectively. These embryos should be surrounded by the embryo sac tissue. On the same medium late globular (0.08–0.1 mm) and early heart-stage embryos (0.1–0.3 mm) developed into plants in moderately high and high frequencies of 48% and 83%, respectively. The presence of the embryo sac at these stages was still beneficial, but no longer a prerequisite. Late heart-stage embryos (0.3–0.8 mm) also showed high frequencies of plant formation, 63%, if Kn was applied at a concentration of 1 mg 1^–1. From the early cotyledon stage onwards, the frequency of plant formation gradually decreased, reaching a minimum at the late cotyledon stage. Subsequently it began to increase again up to the late maturation stage. The poor plant formation shown by the intermediate-aged embryos could be improved slightly by lowering the sucrose concentration to 0.5% and by increasing the Kn concentration to 10 mg 1^–1. Relative to the wild species, embryos of C. sativus showed lower percentages of plant formation. The optimum sucrose concentration was 2% for the heart-stage C. sativus embryos. In all three species the ability to form plants strongly decreased with increasing embryo age, from early to late cotyledon. This is thought to be caused by the increasing tendency of the embryos at these stages to continue in vitro the normal embryo development.     

Sex determination in the dioecious Melandrium

Summary Melandrium album (2n=24), a dioecious species with heteromorphic sex chromosomes (XY, males and XX, females), has a strong genetic commitment for sex determination. We report here a procedure for obtaining haploid plants from cultured anthers and show that genotype, pollen stage, cold treatment and certain culture media components are essential for a reproducible yield of embryos. Our procedure increased the number of responsive anthers and not the number of responsive microspores per anther. Most likely, our experimental system allows the recovery of competent microspores, and this on a medium containing either an auxin or a cytokinin. All of the 36 anther-derived plants tested expressed a female phenotypic sex instead of the theoretical one male one female ratio. When analysed cytologically, the plants exhibited the corresponding female genetic sex (one or two X chromosomes).     

Callus culture ofPterocladia capillacea (Rhodophyta) and analysis of cell wall polysaccharides

Calluses induced fromPterocladia capillacea have been kept in culture for more than three years. They exhibit a fast growth rate, owing to the release of single cells, which in turn develop into new callus. The effect of various media and culture conditions upon growth was investigated. In order to confirm the identity of the callus cells, a 0,45 mg incoculum was grown that yielded 15 g dried callus within six weeks. Polysaccharides from this material (5.5 g) were analysed by¹³C NMR spectroscopy. This produced a spectrum typical of agar and very similar to the one obtained for agar extracted fromP. capillacea plants. However, the callus agar displayed no gel-forming properties, even after alkali modification.author for correspondence     

3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: A new fluorescent dye to detect mycoplasma contamination in cell cultures

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.     

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 10⁷ cells per ml were obtained. The described system is scaleable.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

罗汉果组培苗的栽培研究

本文报道罗汉果组培苗的栽培研究结果,为罗汉果在生产上推广应用组培苗栽培,提供有效的技术措施。     

小麦—天兰偃麦草—黑麦三属杂种花粉植株诱导条件的研究

研究表明,三属杂种处于单核中晚期阶段的花粉最适于诱导形成愈伤组织。低温预处理对促进三属杂种花粉愈伤组织的诱导有一定的作用。利用以马铃薯提取物为基础物质的马铃薯-Ⅱ培养基作诱导培养基,其愈伤组织诱导与分化的频率比目前两个较好的合成培养基要高。同一个三属杂种F_1春、秋播种植株之间在形成愈伤组织的能力上有较大的差异,秋播材料形成愈伤组织的能力明显高于春播材料。F_(?)杂种植株诱导愈伤组织和分化植株的频率均比F_1杂种明显提高。     

Adhesion,proliferation, and adipogenesis in primary rat cell cultures: effects of collagenous substrata,fibronectin, and serum

Summary The effects of collagenous substrata, fibronectin, and fetal bovine serum on the adhesion, proliferation, and adipogenesis of rat stromal-vascular cells are reported. There was no effect on initial stromal-vascular cell-attachment by fetal bovine serum or fibronectin. The number of cells attached to a hydrated collagen-gel was almost twice (P<0.04) the number attached to dried collagen-gel or dried denatured collagen-gel. Total number of cells after 5 days in culture was similar among the collagenous substrata and among the treatments with or without fibronectin in the growth media. Total number of cells increased significantly (P<0.02) with 10% FBS. Adipocytic formation was inhibited by hydrated collagen-gel (P<0.02) compared to dried collagen-gel or dried, denatured collagenous substrata. An interaction occurred between dried, denatured gel and fetal bovine serum so that total formation of adipocytes increased by increasing the level of fetal bovine serum (P<0.07). Adipocytic formation was inhibited by hydrated collagen-gel at all levels of fetal bovine serum. The percentage of cells that converted to adipocytes was significantly lower (P<0.01) on hydrated collagen-gel compared to dried, denatured or dried collagen-gel. Percentage of conversion was not significantly different among levels of fetal bovine serum, although this percentage increased as fetal bovine serum level increased. Adipocytic conversion was not different between fibronectin-treated or untreated cells. Morphology of stromal vascular cells was similar on dried collagen and dried, denatured collagen-gel, but tended to remain bipolar on hydrated collagen-gel. These studies indicate that fetal bovine serum in combination with the extracellular matrix (dried, denatured collagen) increased the differentiation of rat stromal-vascular cells into adipocytes, and that hydrated collagen inhibited differentiation.     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient