Rapid multiplication of Sapium sebiferum Roxb. by tissue culture

Multiple shoots formation and elongation was induced from stem explants of Sapium seedlings on media containing cytokinins. Leaf explants produced callus on a medium containing cytokinins, auxin, casein hydrolysate and coconut milk, which could be induced to form multiple shoots on transfer to a medium lacking casein hydrolysate, coconut milk and auxin. Rooting of isolated shoots by treatment with an auxin mixture (indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid) and transfer of the plantlets to field have also been successful.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Studies on encystment of Echinostoma revolutum cercariae.

Cercariae of Echinostoma revolutum encysted in the kidney of the snail Physa heterostropha within 1 hr and on mucus trails from Helisoma trivolvis, P. heterostropha and Lymnaea sp. within 2 hr. Significantly, more normal cysts were formed in mucus of Helisoma than in mucus of Physa or Lymnaea. Optimal, in vitro encystment occurred within 24 h in either Locke's 1 : 1 or Locke's 1 : 1 + 1% glucose. Significantly more normal cysts occurred in the Locke's 1 : 1 medium. Both normal and abnormal cysts from Lock's media and snail mucus excysted in an alkaline bile trypsin medium. Cercariae did not encyst in Lock'e media supplemented with casein hydrolysate or in agar cultures containing various chemicals.     

Enhanced development of somatic embryos of Plantago ovata Forsk. By additives

Summary Plantago ovata Forsk (commonly known as Isabgul) is an economically important medicinal plant. In the present investigation, in vitro plant regeneration of P. ovata was attempted through somatic embryogenesis. Casein hydrolysate and coconut water were used in different concentrations in Murashige and Skoog medium along with 1-naphthaleneacetic acid and N⁶-benzyladenine to increase the amount of callus and number of somatic embryos. Light and scanning electron microscopic studies followed the developmental stages of embryo formation. Results indicated that optimum concentrations of casein hydrolysate and coconut water are useful for promoting the growth of embryogenic cultures. However, a supra-optimal dose of casein hydrolysate and coconut water induced polyphenol synthesis and caused browning of callus and also eventual death of embryos. The use of additives such as coconut water and casein hydrolysate promotes large-scale production of P. ovata through in vitro somatic embryogenesis.     

Catabolism of Amino Acids by Megasphaera elsdenii LC1

The amino acids in an acid hydrolysate of casein were catabolized more extensively by Megasphaera elsdenii than those in an enzymic hydrolysate. Threonine and serine were most actively degraded, but no resultant increase in growth yield occurred. Branched-chain volatile fatty acid production, which increased as the dilution rate of a glucose-limited chemostat decreased, seemed to be associated with maintenance rather than with growth.     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

Effect of glyphosate on carrot and tobacco cells

The growth of suspension-cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L. cv. Xanthi) cells was inhibited by glyphosate (N-[phosphonomethyl]glycine). This inhibition was reversed by adding combinations of phenylalanine, tyrosine, and tryptophan or casein hydrolysate. Casein hydrolysate and phenylalanine + tyrosine + tryptophan were the most effective treatments. Reversal of glyphosate-induced inhibition occurred only if the aromatic amino acids were added during the first 8 days of glyphosate incubation. Glyphosate uptake was not reduced when the aromatic amino acids or casein hydrolysate were added.     

A Nitrate Reductase-less Variant Isolated from Suspension Cultures of Datura innoxia (Mill.)

A comparative study has been carried out of the growth of two lines of Datura innoxia (Mill.) cells, designated DI-6 and NR1, their resistance to chlorate, and their ability to assimilate nitrate in sterile culture. The NR1 cell line was isolated from DI-6 cultures by first growing the latter in a nitrate-based medium for 5 days and then transferring the cells to a medium containing 2 grams liter⁻¹ of casein hydrolysate as the sole N source and 49 millimolar KClO₃ for a 6-week incubation period. Cells which survived the chlorate treatment then were transferred to casein hydrolysate medium and have been cultured in the absence of chlorate for more than 18 months (NR1).     

Control of Malate Synthase Formation in Rhizopus nigricans

The control of malate synthase formation in a fumaric acid-producing strain of Rhizopus nigricans has been found to be similar in most respects to that of isocitrate lyase, the companion enzyme of the glyoxylate bypass. A basal level is formed in a casein hydrolysate medium, which is repressed by glucose. Utilization of glucose during growth results in relief of glucose repression. Any factor which stimulates growth promotes relief of glucose repression by enhancing the incorporation of repressor metabolites derived from glucose into cell material. Thus, malate synthase formation was enhanced in glucose-containing media by the addition of zinc, or by an increase of the concentration of available nitrogen source in a synthetic medium. Both acetate and glycolate acted as apparent inducers of malate synthase, with glycolate the more effective of the two when added alone. Acetate induction was enhanced by Zn⁺⁺, however, whereas induction by glycolate was unaffected. This supports the concept that acetate stimulates formation of glyoxylate bypass enzymes by a derepression mechanism, whereas glycolate or a product derived from it acts directly as an inducer. Moreover, it is indicated that the malate synthases induced by acetate and glycolate are separate and distinct, as has been shown in Escherichia coli.     

Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect.     

Secretion of Proteinases with Fibrinolytic Activity by Micromycetes of the Genus <Emphasis Type="Italic">Aspergillus</Emphasis>

Proteolytic activity of extracellular enzymes of 11 strains of different Aspergillus species was studied. Comparison of the enzymatic indices of strains grown on agar medium containing either casein or fibrin allowed the selection of the strain Aspergillus terreus 2 as a promising producer of fibrinolytic proteases. It was found that A. terreus 2 proteinases demonstrated maximum activity at pH 8.0. The highest values of fibrinolytic and total proteolytic activities expressed in U_Tyr (amount of micromoles of tyrosine released from fibrin or casein for 1 min) were 34.0 and 358.3, respectively. Maximum activities were detected when growing the producer on a medium containing only amine nitrogen sources (fish flour hydrolysate and peptone); however, the amount of extracellular protein and the specific fibrinolytic and total proteolytic activities were greater in the medium containing both mineral and amine nitrogen sources (fish flour hydrolysate and sodium nitrate) than in the medium containing only fish flour hydrolysate and peptone as nitrogen sources.     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.     

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.     

The effect of fresh toad bile on the induction of encystation in Opalina sudafricana parasitic in Bufo regularis

Opalina sudafricana reacted by encystation to small doses (0.04 ml) of fresh toad bile injected subcutaneously into its host Bufo regularis. It is speculated that androgens present in the injected bile of male toads, and oestrogens found in injected bile of female hosts reach the parasites in the recta of the treated animals and induce them to encyst. High doses of bile killed the parasites and their hosts. Small doses of bile (0·04 ml) added to in vitro cultures induced encystation in the opalinids. High doses killed the opalinids in vitro. There is also the possibility that bufodeoxycholic acid found in the toad bile may play a role in inducing the parasites to divide and encyst.     

Embryoid and Plantlet Formation from Stock Cultures of Nigella Tissues

Embryogenesis could be induced within a short period in differentiated roots of Nigella sativa on solid Murashige and Skoog's medium containing casein hydrolysate (500 mg/1) and IAA (0.5 mg/1). Experimental root explants were taken from two different stocks: (i) previously differentiated from leaf callus and now being maintained through subcultures in liquid White's medium and (ii) freshly differentiated from leaf callus on solid Murashige and Skoog's medium + casein hydrolysate (100 mg/1) + IAA (0.5 mg/1). Fifty per cent of the embryoids produced 2n plantlets within 40–60 days.     

Biological aspects of the interaction between Coelomomyces psorophorae zygotes and the larvae of Culiseta inornata: Environmental factors

Zygotes of the obligately parasitic watermold Coelomomyces psorophorae encyst on the cuticular surface of susceptible mosquito larvae as the initial step in the infection process. Zygotes can successfully encyst on Culiseta inornata larvae at temperatures from 12°–30°C, with an optimum of 25°C. Optimum encystment on larvae occurs from pH 6.5–8.5, with activity documented from pH 5.0–9.5. Zygotes are active in water with total salinity of 5.4 ppt down to double glass-distilled water.     

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.     

Culture Age, Intracellular Ca2+ Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus

In Colpoda cucullus, intracellular Ca²⁺ mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca²⁺ concentration (F₃₄₀/F₃₈₀ ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca²⁺-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.