Stabilization of the synthetic media for plant tissue and cell cultures

In standardMurashige-Skoog medium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na₂EDTA. up to 10% free Fe^II ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na₂EDTA (at the approximate molar ratio Fe^II: Na₂EDTA 1: 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8 – 6.0. We also attempted to increase the relatively low buffering capacity of Murashige-Skoog medium. The addition of sodium citrate (1.25 mmol 1^-1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the Murashige-Skoog medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ± 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelation or of the buffering components.     

Redox Cycling of Iron Supports Growth and Magnetite Synthesis by Aquaspirillum magnetotacticum

Under anaerobic conditions and in the absence of alternative electron acceptors, growth of the magnetic bacterium Aquaspirillum magnetotacticum MSI was iron concentration dependent. Weak chelation of the iron (with quinate, oxalate, or 2,3-dihydroxybenzoate) enhanced growth, whereas strong chelation (with EDTA, citrate, or nitrilotriacetic acid) retarded the growth of strain MSI relative to that of controls lacking chelators. Growth was proportional to the percentage of unchelated iron in medium containing EDTA in various molar ratios to iron. Addition of the respiratory inhibitors antimycin A (5 μM), NaCN (10 mM), and NaN₃ (10 mM) inhibited growth with Fe(III) or NO₃^- as the terminal electron acceptor. Growth with O₂ and NO₃^- was inhibited by 2-heptyl-4-hydroxyquinolone-N-oxide (HOQNO) but not with 2 mM Fe(III). Under strongly reducing conditions, strain MS1 survived but grew poorly and became irreversibly nonmagnetic. Growth and iron reduction in anaerobic cultures were stimulated by the provision of small amounts of O₂ or H₂O₂. Slow infusion of air to cultures which had reduced virtually all of the Fe(III) in the medium (2 mM) supported a high rate of iron reoxidation (relative to killed controls) and growth in proportion to the amount of iron reoxidized. Oxygen consumption by iron-reducing cultures was predominantly biological, since NaCN and HOQNO both inhibited consumption. Inhibition of oxygen consumption (and iron reoxidation) by the addition of ferrozine and the inhibition of iron oxidation (and oxygen consumption) by the addition of HOQNO suggest that iron oxidation by strain MS1 is an aerobic respiratory process, perhaps tied to energy conservation. Iron oxidation was also necessary for magnetite synthesis, since in microaerobic denitrifying cultures, sequestration of reduced iron by ferrozine present in 10-fold molar excess to the available iron resulted in loss of magnetism and a severe drop in the average magnetosome number of the cells.     

Effect of aluminum on iron-induced lipid peroxidation and protein oxidative modification of mouse brain homogenate

In the present study the authors report on the enhancing effect of aluminum(III) (Al[III]) on iron(II)(Fe[II])-induced lipid peroxidation (LPO) of mice brain homogenate, which occurs in a concentration and time-dependent manner. No evidence of LPO caused by Al alone was found. Both Al(III) and Fe(II) ions induced protein oxidative modifications in mice brain homogenate, in a time and concentrationdependent manner. Aluminum enhances Fe(II)-induced protein oxidative modification at a concentration of 2:1 and 1:1 Al:Fe molar ratios. However, Al suppress Fe(II)-induced protein oxidative modification at a concentration of 0.5:1 Al:Fe molar ratio. Addition of ethylenediaminetetraacetic acid (EDTA) inhibits both LPO and protein oxidative modifications induced by Al(III) and Fe(II) ions. Addition of mannitol and of Superoxide dismutase (SOD) did not show such effects. It is concluded that in mice brain homogenate, Al accelerates Fe(II)-induced LPO. Protein oxidative modifications caused by Fe(II) and/or Al ions are enhanced at high, but suppressed at low concentrations of Al ions. The latter observation suggests a possible biological role of Al as an antioxidant.     

Bactericidal effect of Fe2+, ceruloplasmin, and phosphate.

Fe2+, when combined with ceruloplasmin or phosphate, was bactericidal to Escherichia coli at pH 5.0, and when Fe2+, ceruloplasmin, and phosphate were combined, a bactericidal effect was observed under conditions, i.e., short incubation period, in which Fe2+ plus ceruloplasmin and Fe2+ plus phosphate were ineffective. Bactericidal activity increased with the ceruloplasmin or phosphate concentration to a maximum and then decreased as their concentration was further increased. Fe2+ was oxidized in the presence of ceruloplasmin, phosphate, or, in particular, a combination of the two. A bactericidal effect was observed when there was only a partial loss of Fe2+, with more extensive oxidation resulting in a loss of bactericidal activity. The bactericidal effect of Fe2+ plus ceruloplasmin and/or phosphate was unaffected by catalase or superoxide dismutase and was not associated with iodination. Fe-EDTA was also bactericidal at an Fe2+: EDTA molar ratio of 1:0.5, where Fe2+ was partially oxidized. However, in contrast to Fe2+ plus ceruloplasmin and/or phosphate, bactericidal activity was inhibited by catalase and was associated with iodination. Combinations of Fe2+ and Fe3+ were not bactericidal under the conditions employed. A requirement for Fe2+ plus either a product of Fe2+ oxidation or an iron ceruloplasmin and/or phosphate chelate for bactericidal activity is proposed.     

Mobilization of ferritin iron in erythroblasts by chelating agents

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.     

Effects of external iron concentration upon seedling growth and uptake of Fe and phosphate by the common reed, Phragmites australis (Cav.) Trin ex. Steudel

The objectives of this study were to determine whether, and to what degree, the aqueous iron concentration in the growing medium affects the growth of, and Fe uptake by, Phragmites australis, and whether the presence of iron in the growing environment affects the uptake of the essential element phosphate. The wetland macrophyte P. australis was grown under laboratory conditions in nutrient solution (0.31 mg L(-1) phosphate) containing a range of iron concentrations (0-50 mg L(-1) Fe). A threshold of iron concentration (1 mg L(-1)) was found, above which growth of P. australis was significantly inhibited. No direct causal relationship between iron content in aerial tissues and growth inhibition was found, which strongly suggests that iron toxicity cannot explain these results. Phosphate concentrations in aerial tissues were consistently sufficient for growth and development (2-3 % d. wt) despite significant variation in concentration of phosphate associated with roots. External Fe concentration had a significant effect on the growth of P. australis and on both Fe and phosphate concentrations associated with roots. However, neither direct toxicity nor phosphate deficiency could explain the reduction in growth above 1 mg L(-1) external Fe concentration     

Effect of iron (III) in the presence of various ligands on the phagocytic and metabolic activity of human polymorphonuclear leukocytes

FeCl3 or Fe(III) that attached to chelating ligands such as citrate or nitrilotriacetic acid (NTA) at a molar ratio of 1:1 had a toxic effect on PMN. Uptake of radiolabeled Staphylococcus aureus by PMN, preincubated for 2 hr at 37 degrees C in a medium containing Fe(III)-citrate or Fe(III)-NTA, was significantly lower than that of control PMN preincubated without excess iron (p less than 0.002). However, at a 1:2 molar ratio of Fe(III) to citrate or NTA, the iron was not toxic. In contrast, the iron-liganding molecules transferrin and deferoxamine protected the PMN against the noxious effect of iron at concentrations just high enough to sequester all the iron. Fe(III) increased the generation of luminol chemiluminescence by stimulated PMN, whereas the oxygen consumption of the cells was not altered in the presence of Fe(III); this suggests a catalytic effect of iron on the production by PMN of oxygen metabolites at some step beyond the formation of superoxide. No effect of iron was observed when the incubation was performed at 4 degrees C, nor when an oxygen-radical scavenger such as thiourea, mannitol, or catalase was present in the incubation medium. Also, Fe(III) had much less effect on the phagocytic function of PMN of a patient with chronic granulomatous disease. The results indicate that the Fe(III)-induced defect in the phagocytic capacity of PMN depends on the nature and the concentration of the ligand attached to the iron ion, and also suggest that the noxious effect of iron on the PMN function is a result of its ability to catalyze the generation of toxic oxygen species by these cells.     

IRON-MEDIATED CHANGES IN THE GROWTH OF LAKE ERIE PHYTOPLANKTON AND AXENIC ALGAL CULTURES1

The effect of iron enrichment on algal growth and photosynthesis was investigated using natural assemblages of Lake Erie phytoplankton and axenic cultures of Anabaena, Scenedesmus and Selenastrum. Cell yield and photosynthesis were frequently inhibited in the presence of unchelated iron over the range of 3.6 to 53.7 μM iron as FeCl₃. In lake water and in a defined medium with low nutrient concentrations, the degree of inhibition by iron could be reduced by chelating the iron with EDTA or by enriching the cultures with phosphorus. Chemical analyses revealed that the EDTA efectively reduced the ability of the ferric iron to remove soluble phosphorus from the media. EDTA was also observed to reduce rather than enhance iron uptake by axenic cultures of A. flos-aquae. These data support the hypothesis that additions of EDTA to low-nutrient media may serve to stimulate algal growth in the presence of iron by preventing the iron from altering extracellular concentrations of soluble ions essential for algal metabolism. In medium with high nutrient concentrations, the soluble phosphorus concentration was not appreciably altered by either EDTA-chelated or unchelated iron enrichment (0.9 to 53.7 μM). Instead, the observed enhancement of cell yield by EDTA-chelated iron in nutrient-rich media appeared to be due to the direct effect of iron on intracellular metabolic processes.     

Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium composition and substrate feeding

Abstract

Oleandrin has been identified as the most potent antitumor ingredient of the Mediterranean herb Nerium oleander L. A strategy for optimization of medium compositions and conditions was developed for enhanced oleandrin production in suspension cultures from leaf-origin explants of Nerium oleander. The cell suspension cultures were grown in various modifications of MS medium as a basal medium. The effects of different natural extracts, plant growth substances, carbon and nitrogen sources and phosphate on the growth and oleandrin accumulation were investigated as well as effect of light, pH, shaking speed and substrate feeding. The highest oleandrin yield was obtained when the nitrogen concentration was lowered to two-thirds and the phosphate concentration increased by two-thirds of that specified in the MS medium in the presence of 3% sucrose, coconut milk, indolebutyric acid and benzyladenine in concentrations of 1 and 2 mg l^?1, respectively. Lower pH and faster shaking speed favored oleandrin accumulation. Chemical feeding of progesterone and cholesterol boosted the oleandrin concentration to higher levels reaching 8.23±0.05 mg g^?1 dry weight. This was about 10-fold higher than that detected in field-grown plants using the same extraction and analytic conditions, and about 24-fold higher than that determined in control cultures with regular MS medium and without precursor feeding.     

Effect of major nutrients on podophyllotoxin production in <Emphasis Type="Italic">Podophyllum hexandrum</Emphasis> suspension cultures

The effect of major medium ingredients (sugar, nitrogen source and phosphate) in Podophyllum hexandrum suspension cultures was investigated in order to increase the production of podophyllotoxin, the raw material in the synthesis of anticancer drugs. Amongst B5, Eriksson, MS, Nitsch, Street and White's medium, MS medium resulted in high growth and podophyllotoxin accumulation. The optimum level of nitrogen was found to be 60 mM, with a combination of ammonium salts and nitrate in the ratio of 1:2. The highest level of podophyllotoxin was obtained at 60 g glucose/l and at 1.25 mM phosphate after 30 days. Statistical design was adopted to determine the optimum levels of the parameters for cell growth and podophyllotoxin production.     

Studies on the growth and flowering of a short-day plant,Wolffia microscopica

Summary As found earlier, supply of EDTA was obligatory for both flowering and satisfactory vegetative growth in Wolffia microscopica. It is now shown that the metal affecting growth and flowering is most probably iron. Omission of Fe but not of Cu, Zn, Mn and B from the medium markedly affects vegetative growth. There exists also a strong interaction between EDTA and Fe, one being largely inactive in the absence of the other. When Fe-EDDHA is substituted for Fe-citrate and EDTA in the medium, no great effect is seen in vegetative growth, but flowering takes place even under continuous light. Studies with ⁵⁹Fe show that, in the medium containing Fe-EDDHA, Fe uptake is stimulated several-fold; this is apparently associated with the flowering condition.Abbreviations EDTA ethylenediaminetetraacetic acid - EDDHA ethylenediamine-di-o-hydroxyphenylacetic acid     

Nitrogen metabolism in the blue mycelia ofNeurospora crassa isolated from copper toxic cultures

The toxicity of copper on a sole nitrate medium containing KH₂PO₄ as the phosphate source has been studied inNeurospora crassa. Iron counteracted the copper toxicity by suppressing the copper uptake and restored complete growth at a lower iron-copper molar ratio. Nitrite reductase activity was inhibited (75%) in copper toxic cultures, while the nitrate reductase activity was unaltered. Nitrite accumulated in the medium; this indicated decreased conversion of nitrate to ammonia. Alanine transaminase was also inhibited in copper toxicity, resulting in an accumulation of keto acids. Iron could restore the nitrite reductase and the transaminase activities to about 70% of the control value. The accumulation of both nitrite and keto acids disappeared under conditions of reversal of copper toxicity by iron.     

Effects of Humate, Agar-agar, and EDTA on the Development of Tomato Seedlings in Aerated and Non-aerated Water Cultures

The effect of sodium humate on the development of tomato seedlingsin aerated and non-aerated water cultures has been comparedwith the effect of colloidal solutions of agar-agar. Similarcomparisons have been made between the activities of humateand EDTA. The experiments have been conducted in fresh mediaas well as in nutrient solutions that had been formerly usedfor growing other tomato seedlings. Finally, a comparison betweeneffects of humate and EDTA on tomato seedlings growing in nutrientsolutions with various concentrations of calcium, magnesium,and iron salts in aerated and non-aerated water cultures hasbeen carried out. Media used for growth experiments have beentested microbiologically, and qualitative analyses of theirorganic compounds have been made. A distinct relationship between physiological activities ofhumate, EDTA, and the concentrations of calcium, magnesium,and iron cations in the medium has been found. It has also beenshown that in non-aerated media the plants mostly anifer thedeficiency of available iron; this is prevented by the additionof either humate or EDTA.     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

Cell Suspension and Callus Culture from Somatic Tissue of Maize

Cell suspension and callus cultures from somatic tissue of inbred lines of maize (Zea mays L.) were cultured on media that were defined via modification of a Linsmaier and Skoog preparation. Germlings incubated on the primary medium originally employed required long-term incubation for callus induction. Modification of the primary medium with high levels of iron and (ethylene dinitrillo)tetraacetic acid (EDTA), B vitamin amendments and vitamin E, shortened incubation by 75% and nearly doubled the percentage of germlings which produced callus. Callus did not remain viable in subcultures to the secondary medium originally employed, whereas a preparation, developed via modification of the original secondary medium, enabled perpetuation of callus through repeated subculture. Modification with high levels of iron and EDTA, plug B vitamins and vitamin E, with decreased concentrations of five inorganic salts, suppressed aberrant organogenesis and stabilized culture growth as viable callus. Similar modification, with the exception that EDTA was omitted, was employed for the development of a liquid medium. Tonicity of the medium was adjusted with a lowered level of sucrose, with the liquid further modified by addition of acetate. Upon development of this liquid, maize became the sixth monocot species for which somatic cells remain viable through repeated subculture in liquid suspensions.     

Action of iron and iron-complexes onKlebsiella pneumoniae (Klebsiella aerogenes)

Iron in the Fe(III) oxidation state had a negligible effect on the growth ofKlebsiella pneumoniae even at the highest concentration (0.45mm) obtainable without precipitation in a minimal medium containing glucose and inorganic salts together with Tris as the buffer and glycerol 2-phosphate as the phosphorus source. Nevertheless in its presence the toxic action of Cd²⁺, Zn²⁺ and Cu²⁺ was antagonized while that of Co²⁺ and Ni²⁺ was potentiated. Higher iron levels were obtained by supplementing the minimal medium with fructose, glycine, gluconate, tartrate and citrate at a range of concentrations. With fructose and glycine all of the resulting solutions were red-brown and non-toxic. This was also found with the other complexing agents when the ligand:iron ratios were low, but at higher ligand:iron ratios the solutions were green and toxic. Iron-citrate systems were especially toxic but resistance developed and was of the graded type. The results are discussed with particular reference to earlier physico-chemical studies by other workers and it is concluded that the red-brown colour is characteristic of the presence of polymers of high molar mass and that the green colour signifies the formation of low molar mass species.     

Nutrient effect on the biological leaching of a black-schist ore

The purpose of the study was to examine the influence of inorganic N (NH(4), NO(3)) and phosphate on the biological oxidation of a sulfidic black-schist ore which contained pyrrhotite as the main iron sulfide. Iron was initially solubilized as Fe from the ore and subsequently oxidized to Fe in shake flask experiments. Under these experimental conditions, iron dissolution from pyrrhotite was mainly a chemical reaction, with some enhancement by bacteria, whereas the subsequent Fe oxidation was bacterially mediated, with negligible contribution from chemical oxidation. Phosphate amendment did not enhance Fe oxidation. Chemical analysis of leach solutions with no exogenous phosphate revealed that phosphate was solubilized from the black-schist ore. Ammonium amendment (6 mM) enhanced Fe oxidation, whereas the addition of nitrate (6 and 12 mM) had a negative effect. An increase in the temperature from 30 to 35 degrees C slightly enhanced Fe oxidation, but the effect was statistically not significant. The precipitation of potassium jarosite was indicative of Fe oxidation and was absent in nitrate-inhibited cultures because of the lack of Fe oxidation. The black-schist ore also contained phlogopite, which was altered to vermiculite in iron-oxidizing cultures.     

The phytate and mineral content of some cereals,cereal products,legumes, legume products,snack bars,and nuts available in New Zealand

Analyses for phytate by an indirect precipitation method and for the minerals calcium (Ca), zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn) by atomic absorption spectrophotometry were carried out on 100 foods available in New Zealand. Foods with 1% phytate (dry weight basis) included untoasted muesli, rolled oats, wheat germ, wheat bran, soybean, and some soy products. Most breads contained between 0.35 and 0.60% phytate; legumes on average had 0.62% phytate, as did snack bars. There was a wide variation in Ca and Zn contents: There was a tenfold variation in Ca content among the legume products, whereas there was a seventyfold variation in Zn content among the cereals. The phytate: Zn molar ratio, which is presumed to indicate the biovailability of Zn, was above 20∶1 for two-thirds of the cereals and almost all of the snack bars; it was above 15∶1 for one-third of the breads, almost all of the legumes, and half of the legume products. These high phytate: Zn molar ratios, as well as some Ca: phytate molar ratios above 6∶1, indicate that there might be a reduced biovailability of Zn in many of the foods analyzed in this study.     

Characteristics of the freshwater cyanobacterium Microcystis aeruginosa grown in iron-limited continuous culture

A continuous culturing system (chemostat) made of metal-free materials was successfully developed and used to maintain Fe-limited cultures of Microcystis aeruginosa PCC7806 at nanomolar iron (Fe) concentrations (20 to 50 nM total Fe). EDTA was used to maintain Fe in solution, with bioavailable Fe controlled by absorption of light by the ferric EDTA complex and resultant reduction of Fe(III) to Fe(II). A kinetic model describing Fe transformations and biological uptake was applied to determine the biologically available form of Fe (i.e., unchelated ferrous iron) that is produced by photoreductive dissociation of the ferric EDTA complex. Prediction by chemostat theory modified to account for the light-mediated formation of bioavailable Fe rather than total Fe was in good agreement with growth characteristics of M. aeruginosa under Fe limitation. The cellular Fe quota increased with increasing dilution rates in a manner consistent with the Droop theory. Short-term Fe uptake assays using cells maintained at steady state indicated that M. aeruginosa cells vary their maximum Fe uptake rate (ρ(max)) depending on the degree of Fe stress. The rate of Fe uptake was lower for cells grown under conditions of lower Fe availability (i.e., lower dilution rate), suggesting that cells in the continuous cultures adjusted to Fe limitation by decreasing ρ(max) while maintaining a constant affinity for Fe.     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium