GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VII. CELLULAR VARIATION

Variation in long-continued cultures of Haplopappus gracilis and Daucus carota has been investigated. A strain of carrot tissue was isolated that grew with a compact habit, in contrast to the highly friable habit of the parent strain. Its dividing cells were arranged quite differently than in the parent strain. Earlier work had shown that Haplopappus cultures could be reversibly altered in their pigmentation and form, by changing the culture medium. This was confirmed, and it was further shown that pronounced changes in nitrogenous compounds also occurred in response to factors in the medium. However, strains of Haplopappus were isolated which differed persistently from the parent strain, even when they were maintained under the same conditions. The variant strains, grown in the same medium, showed differences in their content of nitrogenous compounds. Stock cultures also changed spontaneously with time with respect to their content of nitrogenous substances. Acriflavine, at low concentration, inhibited the growth and formation of colonics by cells plated on nutrient agar, but, by prolonged exposure to sublethal amounts of the drug, resistant strains were isolated. Certain of the spontaneous variant strains were found to differ from each other and from the parent strain in their chromosome complements in ways that are described and to which the observed changes in morphology and metabolism of the cultures may be attributed. The variations that may occur in the free cells in culture are contrasted with the greater uniformity of the cells as they exist in the plant body.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS IV. THE BEHAVIOR OF THE NUCLEUS

Mitra , J., Marion O. Mapes , and F. C. Steward . (Cornell U., Ithaca, New York.) Growth and organized development of cultured cells. IV. The behavior of the nucleus. Amer. Jour. Bot. 47(5) : 357—368. Illus. 1960.–The nuclei and the chromosomes of carrot cells have been examined at various stages throughout the following sequence: (1) growth of a tissue culture from a preformed explant of secondary phloem from the carrot root; (2) growth and multiplication of carrot cells freely suspended in a liquid medium; (3) growth and re-formation of organs (roots) and whole plants (including flowers) from cells in the freely suspended state. The cells of the carrot are normally diploid (2n = 18), the cells which develop in the explant are also diploid, and the cells of the re-formed organs, and even the flowers developed upon plants grown from cells, are also normal and diploid; normal meioses also occur. Nevertheless, the wide range in growth and form of the freely suspended cells is accompanied by a rich diversity of cytological conditions; these include tetraploid and highly polyploid nuclei which divide, haploidy and such chromosomal aberrations as di- and even tri-centric bridges. Two division figures showing chromosome numbers at different levels of ploidy were seen within the confines of one large cell, and, in another, 2 adjacent division figures were observed with chromosome numbers lower than diploid. Small thick-walled, densely protoplasmic cells divide to form bi- and tetra-nucleate conditions, and in a giant cell a highly multinucleate condition has been seen. Despite this, however, all the regenerated roots and plants yet examined are normally diploid. The implications of these events are discussed.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VI. THE BEHAVIOR OF INDIVIDUAL CELLS ON NUTRIENT AGAR

Freely suspended cells of a long continuously cultured strain of carrot were dispersed in nutrient agar medium in Petri dishes. After inoculation, cells in randomly chosen fields were photographed at low magnification. The same cells were photographed at intervals over the next 23 days. The fate of 678 units, 24% of which grew visibly, was determined from the photographic record. Cells and units displayed a marked degree of individuality. In some units, there was a progressive increase in cell number; in others, after a few divisions, growth ceased; in still other units, cells enlarged without dividing. Although the evidence of cell division in free cells is conclusive, its incidence and maintenance increased with the size of the initial unit. In general, growth started after an “induction period” of about 5 days, but some cells remained apparently unchanged considerably longer after inoculation, before they started to grow. Although single cells of all sizes were observed to grow by one means or another, it was generally the shorter ones that continued to grow into viable colonies. Elongated cells predominated in the carrot strains used; these grew into colonies first by septation, with little or no over-all increase in size, followed by a stage of cell enlargement. Growth by both cell enlargement and division then ensued. During growth by septation within single cells, some derived cells were seen to burst and die. The most frequent divisions were observed during septation when the average cell generation time was 24 hr. Lacking the controls that normally operate in the intact plant body, there was much variation in the forms assumed during growth, a number of which are illustrated. The importance of epigenetic factors, or external controls, that determine the growth of cells in the plant body is stressed.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.     

GROWTH AND DEVELOPMENT OF CULTURED RADISH ROOTS

Excised cultured roots of Raphanus sativus L. cv. White Icicle elongate and produce a few lateral roots but do not increase in diameter. Lateral expansion is effected when both an auxin (indoleacetic acid or naphthaleneacetic acid) and a cytokinin (benzyladenine) are applied at the cut end of the root. The growth regulator effects are apparent first in the pericycle and subsequently in the procambium. Both of these groups of cells divide, producing large numbers of secondary derivatives. The increase in number of cells is reflected in an increase in root diameter. When cultured roots are treated with auxin only, a limited number of pericycle cells divide and lateral roots develop. When roots are treated with cytokinin only, all pericycle cells divide and a multiseriate zone of pericycle-derived cells develops. The procambium is not markedly affected by application of a single growth regulator. The distinct and separable responses of pericycle cells to different regulators suggest that the pericycle can be characterized in a functional, as well as a topographic, sense.     

ULTRASTRUCTURE AND FUNCTION OF GROWTH CONES AND AXONS OF CULTURED NERVE CELLS

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.     

GROWTH INDUCTION IN CULTURES OF HAPLOPAPPUS GRACILIS. I. THE BEHAVIOR OF THE CULTURED CELLS

Blakely , L. M., and F. C. Steward . (Cornell U., Ithaca, New York.) Growth induction in cultures of Haplopappus gracilis. I. The behavior of the cultured cells. Amer. Jour. Bot. 48(5): 351–358. Illus. 1961.—Because of the unusual cytology of Haplopappus gracilis (2n = 4), a study has been made of the growth of its stem tissue in culture. Although growth may occur on a basal medium supplemented in various ways, it was stimulated for present purposes by the use of a basal medium containing casein hydrolysate, coconut milk (2–10% by volume) and naphthaleneacetic acid (0.5 p.p.m.). A definite synergism between coconut milk and naphthaleneacetic acid was demonstrated. The general form of the colonies so obtained responds to the composition of the medium, and certain effects of pigmentation indicate that the biochemistry of the cultured tissue is also a function of the conditions. The Haplopappus cultures were maintained in liquid culture either in the form of free cells or of the small cell clusters to which they readily gave rise. The form of typical cells and cell clusters is described, and stress is laid upon the range of growth forms that are encountered. Variations in suspensions of cells, or small cell clusters, may be investigated by the application of simple microbiological techniques. Haplopappus gracilis is thus a useful material for the further study of growth and morphogenesis by tissue culture techniques.     

DEVELOPMENT OF JUNCTIONAL COMPLEX IN CULTURED CELLS

The development of specialized intercellular junctions in cultured cells was studied ultrastructurally. MDCK cells, derived from dog kidney, were fixed in situ at proper times after replating, and the sections were cut perpendicular to the plane of the monolayer. In three or four days, the apposition of cell membranes and condensation of extracellular flocculent material were observed between the neighboring cells, and such were regarded as the early signs of desmosome formation. In many cases, a desmosome was formed first, and the formation of a tight junction followed on the apical (medium facing) side. Finally, all intercellular spaces were closed by a junctional complex at the apical edge. In the complex, a tight junction, (intermediate junction) and desmosome(s) succeeded each other in a medium-substratum direction in all cases. In glutaraldehyde-O^sO⁴ fixed specimens, the intermediate dense line in the desmosome was ascertained from the infant stage of development, while in O^sO⁴ fixed material, the structure was obscure throughout the observation but side-arm-like projections were more prominent.     

稀有鮈鲫胚后发育和幼鱼生长的初步研究

本文介绍了室养条件下稀有　鲫胚后发育与幼鱼生长的特点。稀有　鲫的胚后发育过程可分为3个阶段共11个时期,在水温24．7—31．8℃的条件下,初孵仔鱼经25—30d即可完成胚后发育。在胚后发育期间,仔鱼、稚鱼及幼鱼呈线性生长,每日增长0．52mm左右。饲养条件对生长和发育均有很大影响。在相同的饲养条件下,采于四川省汉源县、灌县及彭县的亲鱼,其后代在生长上无显著性差异。     

DIVISION AND GROWTH OF SINGLE MESOPHYLL CELLS ISOLATED ENZYMATICALLY FROM TOBACCO LEAVES

Single mesophyll cells isolated enzymatically from tobacco leaves divided and grew into aggregates of several cells in defined media. Morphological changes accompanying the division and the growth suggested that these cells are in the course of dedifferentiation. Some of the potentialities of these cells as an experimental material for developmental and genetical studies are discussed.     

血管紧张素Ⅱ对培养心肌细胞蛋白质和DNA合成的影响

本文利用体外细胞培养技术,观察了血管紧张素Ⅱ对Ｗｉｓｔａｒ乳鼠的心肌细胞和心肌组织中非心肌细胞的促生长作用。结果表明：随ＡｎｇⅡ浓度的升高,心肌细胞直径、总蛋白含量及＾３Ｈ－Ｌｅｕ掺入率均呈剂量依赖性增加,而各组间＾３Ｈ－ＴｄＲ掺入率及心肌细胞数目则无明显变化。相反,ＡｎｇⅡ在引起心肌组织中非心肌细胞（主要是成纤维细胞）的＾３Ｈ－Ｌｅｕ掺入率增加的同时,也引起其＾３Ｈ－ＴｄＲ掺入率及细胞数目的增加     

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division.
The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.     

GROWTH AND DEVELOPMENT OF REPRODUCTIVE AND VEGETATIVE TISSUES OF BOUGAINVILLEA CULTURED IN VITRO AS A FUNCTION OF CARBOHYDRATE

Inflorescence meristems and vegetative tissues, excised from noninduced Bougainvillea ‘San Diego Red’ plants, were cultured in vitro in media containing either 3% fructose, glucose or sucrose as carbon sources. Growth and development of young leaves were equivalent whether sucrose or fructose was used whereas floret initiation on inflorescence meristems was much greater when fructose or glucose was the carbon sources. Brief (1-3 days) exposure of inflorescence meristems to fructose at the beginning of culture and subsequent transfer to sucrose did not increase development over continuous culture in sucrose. Longer exposures (4-7 days) to fructose with subsequent transfer to sucrose did, however, increase the percentage of meristems developing florets, but such treatment did not increase development to the same level as those exposed to fructose for the entire period in vitro. During the first 18 days of culture, growth of meristems in sucrose was linear while that in fructose was exponential. There was no difference in carbohydrate requirements for floret initiation on meristems excised from short-day induced or noninduced plants, suggesting that induction does not enhance the ability of meristems to utilize sucrose.     

THE EFFECT OF pH ON GROWTH, PROTEIN SYNTHESIS, AND LIPID-RICH PARTICLES OF CULTURED MAMMALIAN CELLS

A simple procedure has been established for controlling and measuring the pH of media in which the bicarbonate-carbonic acid system is the predominant buffer. The HCO^-₃ concentration was maintained at 22.5 mM and the H₂CO₃ concentration was varied by equilibrating the media with 0.5 to 40 per cent CO₂ in air. The curve relating extracellular pH to 3 day cell growth was similar for glass-attached HeLa and Chang liver cells. Maximum growth occurred over a pH range of 7.38 to 7.87. Cell growth declined precipitously on the alkaline side and more gradually on the acid side of the optimal pH range. Comparable pH growth curves were also obtained with newly isolated cells from rat liver and skeletal muscle. It was shown that the effect of pH on growth was independent of the CO₂ concentration and that the essential nutrients in the medium were stable over the pH range studied. Although alkalosis depressed the 3 day cell population, cells exposed to a pH of 8.0 to 8.2 grew at the maximal rate for the first 12 to 24 hours. Growth then ceased abruptly and the cells entered a steady state with respect to net protein synthesis. This was followed by cytoplasmic retraction and cell death. Increasing the concentrations of calcium or magnesium in the medium failed to prevent the effects of alkalosis. Moreover, the increase in CO^-₃ concentration of the media and the concomitant decrease in Ca⁺⁺ ion concentration that occur at high pH were eliminated as determining factors in the growth failure and death. While acidosis had a less pronounced effect on the 3 day cell population, its effect on the growth rate was immediate. The increase in cell generation time was proportional to the H⁺ ion concentration. In each of the cell lines studied, acidosis was accompanied by a striking increase in the number of cytoplasmic perinuclear granules. These granules which stain supravitally with Janus green are extracted from fixed cells with lipid solvents. They maintain their identity in cell homogenates and may be isolated from the other subcellular structures by differential centrifugation; at 100,000 g they form a distinct layer at the top of the supernatant fraction. On the basis of their physical and chemical properties, these granules have been called lipid-rich particles. The accumulation of lipid-rich particles in acidosis was independent of the growth rate and the CO₂ concentration.     

VARIATIONS IN GROWTH AND CELL CYCLE TIME OF ARTERIAL SMOOTH MUSCLE CELLS CULTURED AT LOW DENSITY

The population kinetics of cultured rat arterial smooth muscle cells replated at various low densities were studied by direct counting and observation of the cells. Population doubling time decreases with increasing initial density of the culture. These variations in population doubling times depend on both the variation in the percentage of quiescent cells and on the variation of the mean cell cycle time of non-quiescent cells.     

THE GROWTH AND DIFFERENTIATION OF CULTURED BARLEY EMBRYOS

Norstog , Knut . (Wittenberg U., Springfield, Ohio.) The growth and differentiation of cultured barley embryos. Amer. Jour. Bot. 48(10): 876–884. Illus. 1961.—Cultures of excised embryos of barley, Hordeum vulgare L., were made on a number of different media. Growth on White's medium was promoted by adding coconut milk. In the absence of coconut milk, amino acids did not promote growth and differentiation. Embryos as small as 60 μ were successfully grown in vitro. Smaller embryos had the capacity to initiate root and shoot primordia but did not possess the ability to form such embryonic organs as the scutellum and epiblast. Proembryos developed shoots and roots only after a period of irregular growth in which unorganized masses of cells were formed. Multiple centers of shoot initiation were observed in such embryos. The results of the study suggest that, in barley at least, embryonic form may result from an interaction between the embryo and nutritional, spatial and other factors within the ovule.