MAIZE ENDOSPERM TISSUE GROWN IN VITRO III. DEVELOPMENT OF A SYNTHETIC MEDIUM

Straus , Jacob . (U. Oregon, Eugene.) Maize endosperm tissue grown in vitro. III. Development of a synthetic medium. Amer. Jour. Bot. 47(8) : 641–647. Illus. 1960.—The development of a synthetic medium for the growth of endosperm tissue cultures derived from the maize variety, ‘Black Mexican Sweet,‘ is described. Previously, these tissues required yeast extract, casein hydrolyzate, or tomato juice in the medium in order to grow. The growth-supporting activity of these complexes could be attributed to their organic nitrogen content. The effect of juice extracted from fresh tomatoes is enhanced by autoclaving under acid conditions. Presumably this treatment increases the free amino acid content of the tomato juice. One-dimensional paper chromatograms of tomato juice autoclaved under acid conditions indicated the presence of a large amount of free amino acids. Addition of 1.5 × 10^–2 M asparagine to the basal mineral-sugar-vitamin medium (White's medium plus Nitsch's trace-element solution) resulted in better growth than that supported by yeast extract, tomato juice, or casein hydrolyzate. Arginine was ineffective. Glutamine, glutamic acid and aspartic acid (all at 1.5 × 10^–2 M) supported appreciable growth of the tissue but none of them were nearly so good as asparagine in this respect. Thus, a medium containing minerals, sugar, vitamins, and asparagine is capable of supporting excellent growth of maize endosperm tissue cultures.     

Movement of C-compounds from Maternal Tissue into Maize Seeds Grown in Vitro

Uptake from nutrient media into the cob and translocation of various ¹⁴C-compounds from maternal tissue (cob) into developing maize seeds was examined by using caryopsis cultures. Based on relative ¹⁴C concentrations in the cob and the endosperm, it was concluded that the relative efficiencies of movement of amino acids (leucine, phenylalanine, proline), vitamins (thiamine HCl, nicotinic acid), and nucleic acid bases (adenine, thymine) from the cob to the endosperm were 11 to 250 times lower than that of sucrose. Thiamine was unique in that it was concentrated in the embryo at a level that was almost 10 times higher than in the endosperm. The absence of auxotrophic mutants requiring an organic supplement in higher plants (other than thiamine auxotrophs) may be explained by inadequate translocation of these essential metabolites into the mutant zygotes (embryos) to enable their development to mature seeds.     

PRODUCTION OF VITAMIN B12, THIAMINE,AND BIOTIN BY PHYTOPLANKTON1

Some ecologically important phytoplankters released vitamins into culture medium during growth. Skeletonema costatum and Stephanopyxis turris (vitamin B₁₂-requirers) produced both thiamine (vitamin B₁) and biotin when growing with either 12 or 2 ng vitamin B₁₂/liter. Gonyaulax polyedra (vitamin B₁₂-requirer) produced thiamine with 12 ng vitamin B₁₂/liter, and Coccolithus huxleyi (thiamine-requirer) produced vitamin B₁₂ and biotin with 120 ng thiamine/liter, but only biotin with 10 ng thiamine/liter. The amount of vitamin produced by an alga and rate at which it was produced varied with the phytoplankter, the concentration of the required vitamin, and incubation time. Vitamins produced during early and exponential growth were due to excretions, and those produced at stationary growth resulted from excretion and release due to cell lysis. Uptake of the required vitamin by all phytoplankters was greatest during the first few days of incubation. On continued incubation the rate of uptake/cell decreased. In the sea phytoplankters may contribute a major portion of the amount of dissolved vitamins.     

NUTRITIONAL STUDIES OF ASCOBOLUS IMMERSUS

A synthetic medium for vegetative growth and apothecial formation of 2 compatible strains of A immersus has been formulated as follows: KH₂PO₄, 0.5 g; K₂HPO₄, 0.6 g; MgSO₄·7H₂O, 0.5 g; micronutrients, 0.1 ml (Vogel's); asparagine, 5 g (for vegetative growth), or urea, 1 g (for apothecial formation); dextrin (Merck), 20 g; biotin, 5 μg; thiamine, 100 μg; and distilled water, 1 liter. This medium had a pH of 6.2 to 6.7 without adjustment and was satisfactory. For apothecial formation, 20 g Noble agar (Difco) and 2 g cellulose powder (Whatman) were added to the above medium. Apothecial formation was better at 23–24 than at 25–26 C. Light was necessary for apothecial formation. Dextrin, soluble starch, glucose and mannose were satisfactory carbon sources for both vegetative growth and apothecial formation. This fungus could not use lactose, sucrose, sorbose, mannitol, sorbitol or insulin as carbon sources. It could assimilate nitrate in the form of KNO₃. Optimum yields were obtained with asparagine, aspartic acid, or glutamic acid as the source of nitrogen. The optimum nitrogen-carbon ratio for apothecial formation was about 2% dextrin and 0.1% urea. This ratio was given the highest apothecial rating 10. Ascobolus immersus was deficient in the ability to synthesize biotin and thiamine.     

Thiamine requirement in relation to cytokinin in “normal” and “mutant” strains of tobacco callus

Summary High concentrations of kinetin (400–2,000 g/l) permit continuous growth of tobacco callus cultures (Nicotiana tabacum, var. Wisconsin No. 38) in the absence of exogenous thiamine. On the optimum concentration (1,000 g/l) the tissue has been maintained through 21 bimonthly passages without change in vigor or other growth characteristics.The effect of kinetin is general, not mutagenic, because tissue returned to low-kinetin, thiamine-free medium failed to grow.Kinetin-thiamine interactions in cytokinin mutant strains which were grown without cytokinin in light and darkness suggest that the endogenous content of cytokinins may markedly affect the requirement for thiamine and possibly the tissue content of this vitamin and other growth factors.The viability of tissue on low-kinetin media in enhanced by thiamine, but the addition of this vitamin does not eliminate the requirement for a cytokinin.The great divergence in minimum kinetin concentrations required for growth of the tissue in the presence and absence of thiamine indicates that the growth promoting action of cytokinin must be different in the two cases.     

The nature of the requirement of Saccharomyces carlsbergensis for vitamin B6

Saccharomyces carlsbergensis 4228, an organism widely used for determination of vitamin B₆, grows well without this vitamin if thiamine is also omitted from the basal medium, and an inoculum grown in a thiamine-low medium is used. Thiamine inhibits growth when added to such a medium. The thiazole moiety of thiamine, but not the pyrimidine, is also inhibitory, but less so than thiamine itself.Growth inhibition by thiamine is prevented by vitamin B₆. At low concentrations of thiamine, the amount of vitamin B₆ required for growth increases with the thiamine concentration; at concentrations of thiamine above 1 μg./10 ml. the vitamin B₆ requirement for growth remains essentially constant. Since these higher concentrations of thiamine have been used in methods that utilize this organism for determination of vitamin B₆ (1,2), the validity of these methods is confirmed.In the presence of thiamine, growth was also permitted by additions of the thiamine antagonist, neopyrithiamine. In this case, however, the relationship was fully competitive; i.e., the amount of neopyrithiamine required for growth increased regularly with the thiamine concentration. At concentrations considerably higher than those required for growth, neopyrithiamine again inhibited growth, and this inhibition was prevented by an increase in the thiamine concentration. Thus neopyrithiamine acts by lowering the effective thiamine concentration to subinhibitory levels; if excessive amounts are used, it prevents essential metabolic functions of thiamine and itself becomes toxic. The mechanism by which vitamin B₆ prevents thiamine toxicity is not known.The appearance of a requirement for certain growth factors because of inhibitory effects of other metabolically important compounds, rather than because of an intrinsic inability of the organism to synthesize the growth factor, may be much more common than the few recorded instances of this phenomenon indicate.     

Effect of Cullural Conditions on the Growth of Agrostemma githago Cells in Suspension Culture and the Concomitant Production of an Anti-Plant Virus Substance

An extract of cultured Agroxieinma githago L. cells was found to show potent inhibitory activity against plans virus infection. The effects of cultural conditions on the growth of the cell suspension and on the production of the inhibitor were examined. Since the production of substance was dependent on growth. experiments were made to improve growth. The optimum temperature was 26 to 30°C and optimum pH of the medium before autoclaving was between 5 and 7. In a medium of higher osmotic pressure, the water content of the cultured cells was lowered markedly. The growth rate in a small volume of the medium was higher than that in a larger volume at an early stage of the cultivation, but it was not changed by different inoculum sizes. The cells required thiamine and 2,4-D for growth but no other vitamins or growth regulators. The optimum level of 2,4-D was 0.1 mg/l. Higher sucrose concentration in the medium gave higher production of cell mass and of the inhibitor. However, 3% of sucrose was selected as the most economical concentration. For normal cell growth, the presence of both NH₄NO₃ and KNO₃ as nitrogen sources was required. The use of a single nitrogen source caused a long lag period or inhibition of the cell growth. KH₂PO₄ stimulated the growth when in was used in the level of 2.5 to 5 mM. The cell adhesion on the surface of the fermentor sometimes causes trouble in a large-scale cultivation. It was found that reducing the Ca²⁺ level in the medium prevented the cell adhesion and foaming remarkably. Based on the results obtained, a modified medium was established which was excellent for shortening the culture period and for efficient production of the anti-plant virus inhibitor.     

Characterization of maize endosperm-derived suspension cells throughout the culture cycle and enhancement of tissue friability

Batch suspension cultures derived from developing maize (Zea mays L.) endosperm were examined throughout the culture cycle to determine the interaction between the tissue and the medium in relation to sugar transport and the effect of subculturing procedures on growth and friability. The growth rate and friability were improved by increasing the frequency of subculture or by physical screening during transfer. An increase in the conductivity of the medium preceded a decrease in fresh weight associated with tissue senescence. Sucrose in the medium was rapidly hydrolysed, and fructose was depleted more rapidly than glucose. Tissue sucrose concentration expressed on a dry weight basis was higher during the middle of the growth cycle, but hexose, starch, zein, lipid, and soluble protein levels changed very little. The medium pH declined from 5.2 to about 4.5 within one day of subculture. Medium pH changed to 4.5 within one day regardless of initial pH (3.0 to 7.0), indicating regulation of external pH rather than passive acidification. Results are consistent with studies of sugar uptake by these cultures, and indicate that cell clump size can be manipulated without exogenous auxin. The characterization of this tissue line establishes its suitability as a model system for studies of sugar transport and other biochemical events in developing maize endosperm.     

Nutritional studies of histoplasma capsulatum

Summary A chemically defined medium, composed of inorganic salts, glucose, asparagine, cystine, and a vitamin supplement, has been devised for growth of the yeast phase ofHistoplasma capsulatum. Growth in this medium was abundant and compared favorably with that in media containing complex natural material. Conversion of each of the 20 strains examined was accomplished by one or more passages on agar slants of the medium and incubation of the cultures at 37° C. Yeast phase cultures on this medium have been stored for 6 months or more at approximately 4° C without conversion or loss of viability. Of the 20 strains examined for vitamin requirements of the yeast phase, all were partially deficient for thiamine; nine for inositol; five, either partially or completely deficient for niacin; and one, completely deficient for biotin.No specific amino acid was required for growth of the yeast phase, but an organic source of sulfur and one of nitrogen were essential. Cystine and cysteine were equally effective for growth of the yeast phase when supplied on an equivalent sulfur basis and very little difference in growth occurred in media which contained equal amounts of nitrogen in any one of the following compounds: asparagine, glutamic acid, aspartic acid, and proline,Of the 20 strains, all but one, which requires biotin, were capable of continued growth in the mycelial phase when subcultured on an agar medium containing only inorganic salts and dextrose, but growth was improved significantly by asparagine or casein hydrolysate.This investigation was supported in part by a PHS research grant (AI-03524) from the National Institute of Allergy and Infectious Diseases, Public Health Service.     

Effect of water soluble vitamins and their analogues on growth of Candida albicans II. Vitamin B12, thiamine,oxythiamine, neopyrithiamine,substituted pyrimidines and thiazoles

Summary By employing wide ranges in vitamin concentrations in biotin basal mineral synthetic medium, it was demonstrated that vitamin B₁₂ markedly stimulated the growth ofCandida albicans, the organism showing a partial dependency upon this vitamin. Growth inhibition by 5-fluorouracil was reversed non-competitively by vitamin B₁₂, suggesting that B₁₂ has a role in nucleic acid biosynthesis of the organism. Thiamine was growth stimulatory, the organism being partially dependent upon this vitamin as well. Neopyrithiamine and oxythiamine were growth inhibitory in thiamine-free biotin basal mineral synthetic medium although the halves of each inhibitor compound were non-inhibitory. Neopyrithiamine inhibition was reversed by intact thiamine but not by pyrimidine thiamine or thiazole thiamine; while oxythiamine inhibition was reversed by thiamine and pyrimidine thiamine but not by thiazole thiamine, the inference being drawn that oxythiamine selectively blocks utilization of pyrimidine thiamine. Twenty-seven different substituted pyrimidines, thiazoles and related thiamine compounds were all utilizable byC. albicans in thiamine-free basal synthetic mineral medium, the organism presumably synthesizing thiamine when presented with the constituent parts of these thiamine analogues. Substitution of sulfur of the thiazole ring with oxygen, as in -methyloxazolium, failed to produce an inhibitory compound forC. albicans. Acetylthiamine, allithiamine, cocarboxylase, tetrahydrothiamine and dihydrothiamine were equally as growth stimulatory as thiamine.     

Nutrition of the American strain of Gyrodinium cohnii

Summary Only the Woods Hole strain of Gyrodinium cohnii has grown to high densities (4 million cells/ml) in 7 days in a chemically defined medium. The other strains were slow-growing, and preferred seawater media enriched with organics.The Woods Hole strain, at the onset, required histidine, but was trained to grow on ammonia (no histidine strain). The no-histidine strain of G. cohnii utilized NH₄, several amino acids and amines, did not utilize nitrates as N-sources. Though NH₄ supported continued growth, addition of histidine and betaine resulted in higher densities.Glucose, glycerol, and acetate were the best carbon sources; the combination of 2 or 3 C-sources yielded better growth than any singly. G. cohnii is euryhaline and withstood high concentrations of Tris buffer, but was sensitive to temperatures below 20° C.Biotin was needed. Thiamine is probably synthesized but at a low rate: absence of thiamine resulted in indefinite continued slow growth; the addition of thiamine gave dense, rapidly dividing cultures. G. cohnii may perhaps serve for assay of thiamine and biotin in seawater.Supported in part by contract NR 104-202 with the office of Naval Research and by grand G-1198 of the National Science Foundation.     

Effect of nutrients on the growth of a new alpine strain of Haematococcus (Chlorophyceae) from New Zealand

The microalga Haematococcus lacustris is a source of astaxanthin used widely in aquaculture, pharmaceutical, and cosmetic industries. A new strain of Haematococcus (LCR‐26C‐1f) isolated from the New Zealand alpine zone was evaluated in this study. The influence of vitamins, micronutrients, various carbon and nitrogen sources were investigated to maximize biomass production in batch cultures using shake flasks. Supplementation of vitamins consisting of thiamine, biotin, and cyanocobalamin improved the cell density by 40% over the vitamin‐free medium. Out of the individual vitamins tested, thiamine was shown to be necessary to maintain high cell densities. The best nitrogen source tested was nitrate in the form of sodium nitrate_, at a 40 mM concentration. Heterotrophic growth yielded much lower cell densities compared to autotrophic growth. The micronutrients iron and manganese were essential for growth. However, the best growth was obtained using a micronutrient mix that included iron, copper, cobalt, zinc, manganese and molybdenum.     

Growth of Cryptococcus neoformans in a thiamine-free medium

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 g/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.     

Specific Features of Cultivation of Iris ensata Thunb. Callus Tissue

A continuous callus culture was obtained from zygotic embryos of Japanese iris (Iris ensata Thunb.) on Murashige–Skoog medium supplemented with 2 mg/l -naphthylacetic acid and 0.5 mg/l 6-benzylaminopurine (BAP). It was found that successful callusogenesis required isolated embryos at the wax stage of endosperm development. The optimal combination of phytohormones for the growth of callus tissue was 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l BAP. The pigment composition of I. ensata callus tissue was studied. It was demonstrated that subcultivated callus tissue contained red pigments of flavonoid nature. Under stress cultivation conditions, yellow pigments were formed and the content of red pigments increased.     

Growth of Thermus aquaticus and its TaqI endonuclease production

The culture behaviour of Thermus aquaticus was characterized. The response of the bacterium to various carbon (tryptone, glucose, glycerol) and nitrogen sources (yeast extract, NaNO₃, (NH₄)₂SO₄, leucine, thymine, thiamine, glutamic acid) was studied. Amino acids did not support growth, but CASTENHOLZ salt medium supplemented with yeast extract and glucose or tryptone resulted in good growth and production. A suitable medium composition giving the highest biomass concentration and enzyme yield was developed. The simple medium containing TYE-NaCl resulted in the highest biomass concentration, whereas CASTENHOLZ mineral medium supplemented with tryptone and yeast extract gave the highest specific activity and enzyme yield. The effect of inoculum age and size on growth was also investigated in order to improve the yield and process consistency. The use of shake flasks inoculated with precultures at their early or late stationary phase resulted in the same biomass concentration (0.56 ± 0.015 g/l) and similar maximum specific growth rates (0.258 ± 0.003 h^?1). Inoculum sizes between 1 and 2.5 per cent were optimal for cell growth. As the other papers on thermophilic microorganisms, including the T. aquaticus YT-1 strain, gave qualitative information on growth, the results presented here cannot be compared with others on a quantitative basis. TaqI endonuclease was purified using a 5 step protocol including cell disruption, adsorption, precipitation, column chromatography and final dialysis. The enriched fraction had a specific activity of 33,600 U TaqI endonuclease per mg protein.     

Organic Nutrients in the Media for Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on media containing different organic nutrients. Of the sugars tested sucrose was better than maltose, glucose and fructose, and sucrose had an optimum concentration of 3 to 4 %. D-Mannose was significantly less effective than the other sugars. The amino acid mixtures casamino acids (casein hydrolysate) and tryptone increased growth while yeast extract was inhibitory and malt extract without effect. Optimal concentrations were 2 to 3 g · l^-1 casamino acids and 3 to 4 g · l^-1 tryptone. It was to some extent possible to substitute the amino acid mixtures with a single amino acid (glutamine at 300 mg · l^-1). Arginine was inhibitory and asparagine was without any effect. Vitamins proved to be unnecessary although there was a tendency towards increased growth with nicotinic acid and meso-inositol. Purines and pyrimidines were added to the medium but with no effect. Liquid endosperm from coconuts (10 to 15%) increased growth while the liquid endosperm from Aesculus hippocastanum was inhibitory. On the basis of these results a revised medium is proposed for the in vitro propagation of Cymbidium.     

Nutrition of Volvulina Playfair*

SYNOPSIS. Vitamin B₁₂, biotin and thiamine requirements of 10 strains of Volvulina steinii and 1 strain of V. pringsheimii were studied. Vitamin B₁₂ is required for growth of both species, thiamine stimulates growth slightly, and biotin has no discernible effect on growth. The minimum concentration of vitamin B₁₂ giving a growth response in V. steinii, strain SC-2, was 10^?8 g/ml, and maximum growth response was obtained with 1.1 × 10^?7 g/ml. An organic carbon source is required for growth of V. steinii but not of V. pringsheimii. Growth of V. steinii, strain SC-2, occurred in 20 of 21 carbon sources tested. Optimal growth with each carbon source was largely dependent upon pH. Except for pyruvate, acetate, and ethanol, carbon source utilization was light-dependent, and growth in ethanol was reduced in the dark. Isocitric lyase activity was detected in V. steinii grown on acetate medium.     

NUTRITION OF THE GREEN ALGA GOLENKINIA

Defined media were established for four strains of Golenkinia. Strains 929 and 930 require thiamine and vitamin B₁₂, strain 931 requires the latter and is stimulated by thiamine; only strain 320 and a mutant of strain 931 are capable of completely autotrophic growth. The optimal nitrogen concentration is 0.025 m except for strain 931 where it is 0.015 m and the requirement may be met by nitrate, ammonium, or urea. Optimal levels of the other components are: 0.0025 m KH₂PO₄, 0.0005 to 0.001 m MgSO₄, and 50 ml of a stock trace-element solution per liter. Only strain 931 required calcium; however, its addition stimulates the growth of strains 929 and 930. The optimal pH before autoclaving is 6.8. Maximal growth rates of two to three cell doublings per day and yields of 26 to 30 (log₂) cells per ml were obtained. These growth data compare favorably with those for other unicellular green algae.     

Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

Dense Crithidia Growth and Heme Sparing: Relation to Fe,Cu, Mo Chelation*

SYNOPSIS. Heme, intrinsically required by Trypanosomatidae, is unstable, especially in conventional alkaline (pH 7.2–8.0) media. Low solubility of heme in a pH 6.5 basal medium (developed to assay biopterin with Crithidia fasciculata) posed a problem: in media acidified during growth because of glycolysis, heme precipitated, perhaps contributed to acid-limited growth and interfered with densitometric estimation of growth. The remedy was to: replace glucose with less rapidly metabolized mannitol; distribute media in thin layers to promote oxidation of acetate, fumarate, and malate (presumably leaving an alkaline residue); and buffer heavily with histidine + Good zwitterionic buffers, and superimpcse physiological buffering by arginine + asparagine whose catabolism appeared to yield an excess of NH⁺₄ over acid. Thereupon, Fe and Cu deficiencies sharply limited growth in the medium whose main chelators were: (a) 2,3–dihydroxybenzoic + 5-sulfosalicylic acids (which preferentially bind transitional elements at their higher valences; (b) malic and gluconic acids; and (c) histidine. With unconventionally heightened concentrations of Fe, Cu, and Mo (the latter serving as Cu buffer as well as nutrient per se), the hemin concentration could be lowered, widening the margin of safety for heme solubility. Growth then reached 1.4 × 10⁸ cell/ml. This medium may serve to screen for ligands promoting uptake or release of Fe and Cu. The increased growth is a step towards improving the assay medium for biopterin and practical use of Crithidia to assay several B vitamins and essential amino acids for metazoa.