Embryogenesis and plant regeneration of Medicago spp. in tissue culture

Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2–4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced. Respectively: Biotechnology Department, Alberta Research Council, Agriculture Canada, Beaverlodge, Alberta, and University of Alberta, Edmonton, Alberta, Canada     

Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO₄ oxidation/NaB ³H₄ reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO₄ or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO₄ oxidation, about 80% of the cell-associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO₄/NaB ³H₄ in time course studies of cultures up to 8 days in vitro . Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.     

Postmortem Changes in Binding to the Muscarinic Receptor from Human Cerebral Cortex

Abstract: The effects of storage at 4°C on the antagonist and agonist binding properties of the muscarinic acetyl-choline receptor from fresh surgical and frozen autopsy samples from human cerebral cortex were studied. The number of 1-[³H]3-quinuclidinyl benzilate binding sites and their affinities were stable up to 51 h, both when stored as pieces of intact nonfrozen tissue and as a homogenate. The agonist binding properties as measured by the ability of the muscarinic agonist carbachol to compete with l-[³H]3-quinuclidinyl benzilate were also stable up to 51 h when the tissue was stored in the form of pieces. The affinity for carbachol decreased when the tissue was stored as a homogenate. The frozen autopsy samples showed no significant differences in binding properties in comparison with fresh neurosurgical tissue.     

The interaction of phagocytes and the large-sized parasite Cryptococcus neoformans: Cytochemical and ultrastructural study

Summary The yeast Cryptococcus neoformans may develop under certain conditions a large polysaccharide capsule 50–100 M in diameter and therefore cannot be phagocytosed by either polymorphonuclear cells (PMN's) or mononuclear phagocytes (MN's). The cellular defense mechanism — in various animals — against the yeast is composed by formation of ringlike structure of PMN's or MN's cells which surround the C. neoformans. Ring structures develop either in vivo or in vitro in tissue culture; destruction of the yeast occurs within 36–72 hours.Several hydrolases, such as acid phosphatase, -glucuronidase and non-specific esterase were found to be released from the phagocytic cells into the enclosed yeast. Considerable reduction of NBT used as a marker for oxidative activity was observed in MN rings at contact regions of the MN cells and the yeast. Electron microscopic studies indicate that the phagocytic cells in the ring structure have many pseudopodes penetrating into the polysaccharide capsule of the yeast. Disintegration of the capsule was observed as well as phagocytosis of its material. A possible analogy between normal phagocytosis of small-sized bodies and the ring structure obtained when large bodies are involved is discussed.     

An investigation of ageing in human costal cartilage

Summary Changes in human costal cartilage with increasing age (2–81 years) have been studied in the optical and electron microscope using routine and histochemical techniques.Concurrent with increasing age, chondrocytes undergo degeneration which is characterized initially by the accumulation of lipidic material within cells and, subsequently, by the formation of a halo around degenerating chondrocytes. The halo material is composed of electron dense bodies, amorphous material, and collagen fibrils. Both electron dense bodies and the amorphous material are of cellular origin and they have similar histochemical responses.Using histochemical techniques in the optical and in the electron microscope, it has been shown that chondroitin sulfate decreases with increasing age, while a hyaluronidase resistant material (presumably keratan sulfate) increases, initially in the central zone, and subsequently in the peripheral zones. Hyaluronidase resistant material is minute or absent in the central zone of aged cartilage.The genesis of collagen fibrils progresses from thin unbanded collagen-like fibrils in the pericellular lacunae of chondrocytes in young specimens to thick fibrils (sometimes in excess of 0.5 ) with a period of 640 Å in ageing cartilage. Aggregation of collagen fibrils seems to be related at least initially to the preponderance of matrix granules and beaded filaments which have been shown to originate intracellularly in vacuoles formed in degenerating mitochondria. Both of these structures contain glycosaminoglycans and, with increasing age, glycosaminoglycans decrease while collagen fibrils aggregate. In old age, the amorphous material, and possibly the content of disrupting electron dense bodies, seem to give origin to some collagen fibrils. This and other mechanisms of formation of collagen fibrils have been observed and they are discussed.Calcification of the matrix increases with increasing age and this agrees with previous findings.Supported by grants from the Italian National Research Council. — The authors are indebted to Miss Giuliana Silvestrini and to Mr. Lucio Virgilii for their expert and extensive technical assistance. — To Dr. A. Ascenzi, Director 1° Istituto di Anatomia e Istologia Patologica, and to Dr. C. Cavallero, Director, 2° Istituto di Anatomia e Istologia Patologica, Università di Roma, the senior author would like to express his appreciation for the use of equipment and facilities pursuant to this investigation, while on sabbatical leave from the University of California, Irvine, College of Medicine. — We wish to extend our thanks to the Italian National Research Council for supporting this study.On sabbatical leave from the University of California, Irvine, College of Medicine.     

Fine structure and histochemistry of “calcifying globules” in epiphyseal cartilage

Summary The cartilage matrix in which the early calcium salts are deposited has been studied in the tibial epiphyses and in the costo-chondral junctions of 30-day-old guinea pigs. The results may be summarized as follows:(1) Structures of globular shape (globules) are to be found throughout the entire epiphyseal plate. (2) They have a homogeneous matrix and are bounded by a membrane. (3) Early calcification occurs in globules. Calcification of collagen fibrils seems to occur later. (4) The earliest mineral deposited would seem to consist of tiny granules about 20 Å in diameter. Then apatite crystals are laid down, initially in small clusters and later filling the globules completely. (5) The globules are strongly osmiophilic. They seem to contain a fair amount of neutral polysaccharides, but no acid polysaccharides except a coating on their outer membrane. Hyaluronidase digestion does not affect globules. Papain digestion makes them more reactive to uranium and lead. (6) Globules are of cellular origin but they are almost certainly not pre-formed in the chondrocytes. Finally, the present paper advances the hypothesis that some globules derive from degenerating chondrocytes and others from the processes of normal chondrocytes.The author is indebted to Mr. A. Benvenuti for his technical assistance. This work was supported by a grant from the Italian Research Council.     

基因型和胚龄对小麦未成熟胚离体培养反应的影响

本文对34种基因型的小麦未成熟胚在离体培养中的反应进行了比较。结果表明,94％的供试基因型愈伤组织诱导率都可达到80％以上,若排除供体植株环境条件的不同和接种过程中的人为因素可能造成的影响,不同基因型的愈伤组织诱导率看来没有根本的差异。愈伤组织分化率因基因型的不同变动在0—60％之间,平均为32.7％。虽然同一基因型的盾片愈伤组织分化率在不同年份中有所不同,但是愈伤组织是否具有再生能力?看来是个稳定的遗传性状。因此小麦未成熟胚对愈伤组织诱导的反应和愈伤组织的再生能力可能具有不同的遗传基础。本文的结果还表明,虽然最适于培养的未成熟胚的大小为1毫米左右,伹小至0.3毫米的未成熱胚仍能以几乎100％的频率形成愈伤组织,60％左右的愈伤组织能分化出再生檀株,只是所需的时间比1毫米左右的胚较长。     

Glutamate Stimulation of [3H]Dopamine Release from Dissociated Cell Cultures of Rat Ventral Mesencephalon

In dissociated cell cultures of fetal rat ventral mesencephalon preloaded with [3H]dopamine, glutamate (10(-5)-10(-3) M) stimulated the release of [3H]dopamine. Glutamate stimulation of [3H]dopamine release was Ca2+ dependent and was blocked by the glutamate antagonist, cis-2,3-piperidine dicarboxylic acid. Glutamate stimulation of [3H]dopamine release was not due to glutamate neurotoxicity because (1) glutamate did not cause release of a cytosolic marker, lactate dehydrogenase, and (2) preincubation of cultures with glutamate did not impair subsequent ability of the cells to take up or release [3H]dopamine. Thus, these dissociated cell cultures appear to provide a good model system to characterize glutamate stimulation of dopamine release. Release of [3H]dopamine from these cultures was stimulated by veratridine, an activator of voltage-sensitive Na+ channels, and this stimulation was blocked by tetrodotoxin. However, glutamate-stimulated [3H]dopamine release was not blocked by tetrodotoxin or Zn2+. Substitution of NaCl in the extracellular medium by sucrose, LiCl, or Na2SO4 had no effect on glutamate stimulation of [3H]dopamine release; however, release was inhibited when NaCl was replaced by choline chloride or N-methyl-D-glucamine HCl. Glutamate-stimulated [3H]-dopamine release was well maintained (60-82% of control) in the presence of Co2+, which blocks Ca2+ action potentials, and was unaffected by the local anesthetic, lidocaine. These results are discussed in terms of the receptor and ionic mechanisms involved in the stimulation of dopamine release by excitatory amino acids.     

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N⁶-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday