MORPHOGENETIC STUDIES ON HAWORTHIA: EFFECTS OF INOSITOL ON GROWTH AND DIFFERENTIATION

Effects of inositol on growth and differentiation of Haworthia tissue were studied. Inositol was found to be essential for the survival of the tissue. Growth was increasingly better with the increase in inositol content of the medium. A minimum level of inositol was required for organ differentiation. Organ differentiation was also promoted by the increase in inositol concentration in the medium. The chlorophyll content of the tissue increased with the increasing concentration of inositol. Presence of 2-deoxyglucose in the inositol-containing medium led to inositol deficiency symptoms.     

Carbohydrate supplements to the callus culture medium modify the growth of potato tuber moth larvae feeding on Lycopersicon chmielewskii callus

Lycopersicon esculentum and L. chmielewskii are respectively susceptible and resistant to the potato tuber moth (Phthorimaea operculella Zeller) in the field. Feeding bioassays were conducted with the herbivore caterpillars reared on callus derived from both tomato species and grown in vitro, and the influence of carbohydrate supplements to the callus culture medium, on the insect's feeding behavior was investigated. Newly-hatched larvae fed with L. esculentum or L. chmielewskii callus raised on a medium with 88 mM sucrose, reached a weight of 12–15 mg and 1.5–3.0 mg, respectively, within 9 days. Restriction of larval weight increase in insects reared on L. chmielewskii callus, disappeared when the host tissue was transferred 24 h prior to the callus-insect assay to a medium supplemented with 264 mM of either sucrose, glucose, fructose or mannose. The capability of L. chmielewskii callus to restrict growth of larvae was restored in host tissue retransferred from a medium with 264 mM sucrose to a 24-h incubation on one supplemented with 264 mM of either mannitol, sorbitol, glycerol or myo-inositol, before the callus-insect bioassay. The larval growth response remained unaltered by callus incubated on a medium with 264 mM xylose. The ameliorating effect on insect growth of high sucrose in the callus medium was not due to sucrose as an ingredient of the insect's diet. The diverse response of L. chmielewskii callus, and its dependence on the type of carbohydrate in the medium, rule out effects of these substances as nonspecific medium osmotica. The swift callus responses to carbohydrates (within hours of a change in medium composition), as reflected in the insect's growth, were not accompanied by visible morphological variations in the host tissue. We suggest that suppression by high levels of exogenously applied saccharides and derepression by exogenous polyols and myo-inositol of the impedement to growth of the potato tuber moth larva, reflect the existence in L. chmielewskii of a carbon metabolic control mechanism of gene expression whose products affect insect growth.     

Chlorophyllous Nature and Growth Characteristics of Teratoma and Habituated Tissue Cultures of Tobacco

The growth characteristics of three types of tissue cultures of tobacco have been investigated. They are (i) pigmented and differentiated teratoma tissue; (ii) pigmented and non-differentiated habituated tissue and (iii) non-pigmented and non-differentiated habituated tissue. All the tissues were grown on a 10 × White's medium without any exogenous supply of auxin. The comparative growth rate of the tissues was related to their nature of differentiation, viz., either organelle differentiation with chloroplast or organ differentiation with buds, leafy appendages, etc. Teratoma and pigmented habituated tissues had normal chlorophyll and carotenoid contents and showed more favorable growth in light than in the dark. The pigmented tissues showed a higher protein content than the non-pigmented tissues irrespective of whether they were of tumorous or normal origin. The pigmented tissues showed less dependence on sucrose availability in the medium than the non-pigmented tissues. Recovery after sucrose starvation was higher in the pigmented tissues than in the non-pigmented tissues. The results indicate that the growth rate of highly differentiated tissues is much slower than non-differentiated tissues and that the pigmented tissues photosynthesize and survive on reserve food products manufactured as a result of photosynthetic activity.     

STUDIES ON STRIGA SENEGALENSIS III. IN VITRO CULTURE OF SEEDLINGS. ESTABLISHMENT OF CULTURES

Striga senegalensis seeds were cultured on Knop's and modified Murashige-Skoog's medium. An aqueous root exudate/extract of the host (Sorghum vulgare) was found essential for the germination of seeds in vitro. The seedlings grew better on a medium containing sucrose than on mineral salts alone. Only limited growth was achieved on Knop's medium. Better growth was supported by Murashige-Skoog's medium. By transfer to larger culture vessels seedlings were kept in the latter for several months. The seedlings developed chlorophyllous shoots in light and flowered after 4–4½ months. The successful culture of these seedlings in a simple medium of only mineral salts and sugar indicates that the parasitic seedling depends on the host for only water, mineral salts and sugar and not for more elaborate substances.     

THE BEHAVIOR OF TISSUE CULTURES FROM ENGLISH AND ALGERIAN IVY IN DIFFERENT GROWTH PHASES

In both the English and Algerian ivies, Hedera helix L. and Hedera canariensis Willd., leaf dimorphism of the juvenile and the mature, fruiting growth phases is pronounced, the former being a vine with lobed leaves and the latter a shrubby, upright form with entire leaves. Tissue cultures of English ivy started from stems of the two growth phases on the same plant consistently behaved differently, those from the juvenile stage invariably having the higher proliferation rate and larger cells. These differences were maintained consistently in monthly transfers over a period of two years. No medium was found which would support the growth of tissue subcultures of the adult stage of Algerian ivy, but all growth phases of the English ivy were cultured freely in a modified White's medium with additions of coconut water, naphthaleneacetic acid, enzymatic casein hydrolysate, and inositol. Cultures from individual open-pollinated seedlings of both species varied greatly in proliferation rate but were usually high.     

In vitro Studies on Pinus

Hypocotyl tissue from Pinus gerardiana was established in culture on White's basal medium supplemented with 2 % sucrose, 10% (v/v) coconut milk, 500 mg/l casein hydrolysate and 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). Various organic and inorganic nutrients were studied in order to determine the specific nutritional requirements of the tissue in vitro. Sucrose, glucose and maltose were equally effective as fixed carbon sources. The inorganic nutrient combination of White's medium was found to be better than that of Murashige and Skoog's medium. White's modified basal medium supplemented with coconut milk, casein hydrolysate and 2,4-D was the most successful nutrient combination. Glutamine was as effective as casein hydrolysate in promoting growth of the tissue.     

INDUCTION OF XYLEM ELEMENTS IN ISOLATED COLEUS PITH

Small pith explants from the fifth internode of Coleus blumei were placed on a defined medium supplemented with different concentrations of indoleacetic acid (IAA). With all levels of IAA tissue growth was slight; however, when the medium contained more than 10^-7 m IAA, xylem elements developed after 11-14 days. Omission of sucrose from the medium prevented this differentiation of xylem elements. Isolated xylem cells or small nodules were most common, but long strands were also seen. The merits of the Coleus system for study of plant-cell differentiation are discussed.     

GROWTH STUDIES OF THREE CHLOROPHYLLOUS CALLUS PHENOTYPES OF GLYCINE MAX

The establishment of three chlorophyllous callus phenotypes, Glycine max (L.) Merrill, cultured on a modified Miller's medium is described. Experiments were designed to determine the hormonal requirement necessary to maintain an adequate callus growth rate that would allow for the phenotypical accumulation of chlorophyll in all phenotypes. Addition of α-naphthaleneacetic acid and kinetin, both at 1 mg/liter, to the basal medium fulfilled this requirement. However, callus growth for these phenotypes required only an exogenous supply of cytokinin. All callus phenotypes, when maintained on 3% sucrose, were shown to possess similar growth curves; however, optimal growth rates of these cultured phenotypes occurred on different levels of exogenous sucrose (NG, 2%; LG, 1%; Y, 2%). Sucrose (filtered and autoclaved) and, in most cases, fructose (filtered), when employed as a carbon source in the basal medium, maintained adequate growth rates. Glucose (filtered) supported only minimal callus growth. These callus phenotypes, after two years in culture, showed a certain degree of cell type differentiation as indicated by the formation of isolated tracheoids and in some cases organized tracheoid development. The chromosome complement (2n = 40) was observed to be polyploid.     

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.     

A novel technique to propagate primary human preadipocytes without loss of differentiation capacity

Objective: The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity. Research Methods and Procedures: Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor‐2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol‐3‐phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages. Results: The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 µM insulin, resulted in lower doubling times at all seeding densities tested (0.05 × 10⁴ to 1.5 × 10⁴) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol‐3‐phosphate dehydrogenase activity, 493 ± 215 vs. 41 ± 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium. Discussion: The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.     

Excised Tobacco Pith Bioassays for Root-knot Nematode-produced Plant Growth Substances

Root-knot nematodes, Meloidogyne incognita, were grown in tobacco pith tissue cultured in vitro on modifired white's medium. The nematodes did not secrete detectable amounts of auxin or cytokinin, but did promote growth in the presence of both types of growth substance. When both IAA and kinetin were supplied, the nematode-infected tissue was larger, more irregularly shaped than control tissue; nematodes induced syncytia and developed past the second larval stage. When either IAA or kinetin was omitted from the medium, nematodeinfected and control tissue failed to grow and the nematodes failed to induce syncytia or develop.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

ROLE OF ASYMMETRIC CELL DIVISION IN PTERIDOPHYTE DIFFERENTIATION. II. EFFECT OF CA2+ ON ASYMMETRIC CELL DIVISION,RHIZOID ELONGATION,AND ANTHERIDIUM DIFFERENTIATION IN VITTARIA GEMMAE

Gametophytes of Vittaria graminifolia reproduce vegetatively by means of gemmae. Each gemma consists of a linear array of six cells: four body cells and a knob-shaped terminal cell at each end. When gemmae are shed from the gametophyte onto Knop's mineral medium, the two terminal cells do not divide, but elongate to form primary rhizoids. The body cells undergo asymmetric cell division, and the smaller daughter cells differentiate into either secondary rhizoids or prothalli. When gibberellic acid is included in the medium, antheridia are formed as a result of asymmetric cell division instead of vegetative structures. We studied the effect of Ca²⁺ on asymmetric cell division, rhizoid elongation, and antheridium formation in gemmae cultured on Knop's mineral medium and variations of Knop's medium. Ca²⁺ inhibited the onset of cell division and rhizoid elongation, but was required for differentiation of antheridia. Treatments which lowered the Ca²⁺ content of gemmae (EGTA and dilute HCl extraction, culture on verapamil-containing and Ca²⁺-deficient medium) caused an early onset of cell division and rhizoid elongation. The stimulation of growth was most pronounced when gemmae were deprived of Ca²⁺ during the first 24 hr of culture. The proportion of cell divisions which differentiated into antheridia in response to GA was greatly reduced when the Ca²⁺ status of gemmae was lowered with verapamil and Ca²⁺-EGTA buffers.     

GROWTH FACTOR INTERACTIONS IN THE TISSUE CULTURE OF TUMOROUS AND NONTUMOROUS NICOTIANA GLAUCA-LANGSDORFFII

Schaeffer , Gideon W., Harold H. Smith and Marion P. Perkus . (Brookhaven Natl. Lab., Upton, N. Y.) Growth factor interactions in the tissue culture of tumorous and nontumorous Nicotiana glauca-langsdorffii. Amer. Jour. Bot. 50(8): 766–771. Illus. 1963.—Tissues representing tumorous and nontumorous Nicotiana glauca-langsdorffii were cultured on high (5 ×) and low (1 ×) concentrations of a modified White's basal medium containing 2.9 × 10^–6m indoleacetic acid. The growth responses of tissues of both the tumorous and nontumorous genotypes to supplements of kinetin, glutamine, inositol and nucleic acid constituents added singly and in all combinations were noted on high-salt media. The nucleic acid components inhibited growth and were omitted from low-salt media. The best growth response was observed with glutamine and inositol for tissues from the tumorous hybrid and with glutamine, inositol and kinetin in the nontumorous type. Kinetin was a distinct and consistent requirement for rapid growth of nontumorous tissues, but no appreciable kinetin effect could be observed with tissues from the tumorous genotype.     

COMPETITIVE EXCLUSION AMONG THREE PLANKTONIC BLUE-GREEN ALGAL SPECIES1,2

Although domination of 1 species in an algal community has often been attributed to toxic effects, nutrient exhaustion can also lead to competitive exclusion among algal species. The bacteria-associated blue-green algae Microcystis aeruginosa, Nostoc muscorum, and Phormidium foveolarum were cultivated in Zehnder-Gorham's medium No. 11 individually and in their combinations. The growth rates of Microcystis and of Nostoc were reduced by the presence of Phormidium. This detrimental effect was apparently not due to formation of any toxic or allelopathic matter. It was caused by the reduction of iron and trace metal concentrations in the medium by Phormidium to levels too low for normal growth of Microcystis and Nostoc. Work with the axenic forms of the 3 algal species also indicated a lowering of iron and trace element concentrations by Phormidium to levels not sufficient for sustained growth of Microcystis and Nostoc. Thus, bacteria played no significant role in this example of competitive exclusion.     

Effects of ionizing radiation (60Co gamma rays) on growth and morphogenesis ofHaworthia mirabilis,haw. Callus tissue

Summary The effects of γ-radiation on growth and morphogenesis ofHaworthia callus in vitro were determined. The doses ranged from 100 to 5000 rads. Survival, growth pattern, growth rate, and differentiation of vegetative buds and roots in both irradiated and nonirradiated callus were compared. Growth data up to 24 weeks for irradiated and control cultures were analyzed. The dose range between 800 to 2500 rads produced compact callus as compared to the controls which were friable. After 12 weeks all control cultures differentiated vegetative buds with roots, whereas callus exposed to 800 to 2500 rads continued to grow with little or no organogenesis. However, it was observed that the wet and dry weights of callus receiving 1000 to 1500 rads ultimately exceeded those of nonirradiated controls.     

DROUGHT STRESS RESPONSES OF MICROSERIS SPECIES DIFFERING IN NUCLEAR DNA CONTENT

Anatomical and physiological responses to drought stress were compared in two Microseris species differing in DNA content and originating from contrasting habitats relative to water availability (M. bigelovii, DNA = 2.6 pg nucleus^–1, more xeric; M. laciniata, DNA = 6.8 pg nucleus^–1, mesic). Leaf mesophyll cell volume was positively correlated with DNA content and negatively correlated with tissue elasticity, i.e., low ϵ̄ and thin cell walls. Drought stress increased leaf tissue elasticity (lower ϵ̄, thinner cell walls). Cell volume, cell wall thickness, cell number, and leaf area were decreased most by drought stress in M. laciniata. Osmotic adjustment with a 20% increase in total solutes (mostly amino acids) after stress was observed in both species, but their estimated contribution to the change in osmotic potential was larger in M. bigelovii. These findings indicate that the Microseris species studied respond to low water availability by maintaining turgor with 1) small cell volumes, 2) elastic tissues (low ϵ̄, thin cell walls), and 3) osmotic adjustment. Both enhanced tissue elasticity and small cell volume appear to be inherent characteristics in M. bigelovii and drought-induced responses in M. laciniata. These data are compatible with the hypothesis that natural selection may influence DNA content through differential sensitivity of cell growth to environmental stress.     

Axenic Cultivation of Phytomonas davidi Lafont (Trypanosomatidae), a Symbiote of Laticiferous Plants (Euphorbiaceae)*

SYNOPSIS. Phytomonas davidi (Trypanosomatidae) originally discovered by Lafont in 1909 on the island of Mauritius was rediscovered in Euphorbia cyathophora in Florida. Successful cultures were established in diphasic medium consisting of duck blood agar and modified Phillips’medium as overlay. Optimal growth was obtained when Mansour's medium was used as overlay and poorest growth when Cowperthwaite's medium buffered at pH 5.0 was utilized for this purpose. Marked changes tending toward choanomastigotes rather than the elongate twisted promastigotes were observed in cultures.     

Patterns of root activity and responses of species to nutrients in vegetation of fertile alluvial soil

Temporal pattern of growth, time and depth of root activity, and responses to N, P and K enrichment were measured for the three most abundant species in species-poor vegetation on fertile alluvial soil to examine whether differences in these characteristics could account for their co-existence. The responses of these co-existing species to nutrients were tested in a factorial experiment of N, P and K additions. In the control plots, repeated harvests and injections of Sr at different depths in the soil were used to test differences among species in temporal and spatial pattern of root activity. Root activities were assessed from the Sr concentrations in the aboveground biomass. Differences in temporal pattern of growth and root activity, but not differences in spatial root activity between species, could account for the co-existence of species, since Conium, the most abundant species, was the earliest grown and the deepest-rooted species and it died back when the other two species started to maximize their growth and root activity. In comparison with the second most abundant species Lactuca, Conium had higher N but lower P tissue concentrations. Addition of N favoured Conium and almost eliminated Lactuca, while P and/or K additions increased the abundance of Lactuca and restricted that of Conium. These results provide indications that the differential responses of species to nutrients could be explained by species co-existence also in fertile soils. The changes in vegetation composition after nutrient enrichment could merely be predicted by the species' tissue concentrations of nutrients. The addition of a particular nutrient tended to favor the species with the highest tissue concentration of this nutrient.