Partial Synchronization of Carrot Cell Culture by Auxin Deprivation

A strain of carrot cells (Daucus carota cv. Kintoki) grew exponentially in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/1) with a doubling time of about 2 days. When those cells were transferred to a medium lacking 2,4-D, they continued to grow at almost the same rate for about a week. When the cells were again transferred to the auxin-free medium, the rate of cell division gradually decreased. After the cell division had ceased, cells were returned to the ordinary 2,4-D medium. A burst of cell divisions occurred after about 2 days. Timing of DNA synthesis and of mitosis suggested that the cells had been arrested at G₁ phase. In a medium containing indoleacetic acid instead of 2,4-D, the auxin was rapidly degraded and the culture was similarly synchronized as in the auxin-omitted medium.     

The Effect of 2,3.5 Triiodobenzoic Acid on Caulogenesis in Callus Cultures of Tomato and Pelargonium

Stem explants from winter grown tomatoes cultured on a cytokinin, auxin-free medium, developed one or two adventitious shoots at the top end of the explant. Addition of the auxin transport inhibitor. 2,3,5-triiodobenzoic acid (TIBA) to the medium stimulated caulogenesis with loss of polarity. Callus, initiation in pelargonium and ‘geranium’ petiole explants requires both auxin and cytokinin. On transfer, after callus induction to an auxin-free medium, rhizogenesis occurs in pelargonium cultures followed by caulogenesis. Few shoots develop and unless these are removed, further caulogenesis is suppressed. Bud-like structures were formed in the callus. Subculture on auxin-free medium containing cytokinin and TIBA resulted in shoot formation from these bud-like organs. An analogy with apical dominance is suggested. In ‘geranium’ callus, shoots developed with a low frequency (c. in 2% of the cultures): caulogenesis was increased to 80% when calli were subcultured from auxin-free, cytokinin medium after green nodule formation to cytokinin-TIBA medium. Histological studies of green nodules in ‘geranium’ callus indicated a variation in morphological development within and between nodules. It is suggested that auxin synthesis may occur at some microscopic stage in morphogenesis in ‘geranium’ cultures which suppresses further caulogenesis. This may be overcome by the addition of TIBA to the medium at the appropriate stage in morphogenesis. The possible interaction of endogenous auxin in morphogenesis is discussed.     

Protoplast formation,L-colony growth,and regeneration ofClostridium beijerinckii NRRL B-592 and B-593 andClostridium acetobutylicum ATCC 10132

Summary Protocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques.     

Characterization of Odorous Compounds in Rotten Blue-green Algae

An ice-nucleating bacterium, strain KUIN-1, was isolated from the leaves of field beans (Phaseolus vulgaris L.). Strain KUIN-1 was identified as Pseudomonas fluorescens from its taxonomical characteristics. Ice-nucleating activity was obtained when strain KUIN-1 was cultured aerobically in a medium containing Koser citrate broth (pH 7.0) for 24 hr at 18°C. The ice- nucleating activity did not appear until the bacterial cell concentration reached 10⁷ to 10⁸/ml. Nucleation at — 3.0°C was detected in suspensions (1.8 × 10⁹ cells/ml) of cells that had been grown on the medium containing Koser citrate broth. Strain KUIN-1 produced a lower nucleation frequency (i.e. the number of ice nuclei/cell) than did ice-nucleating Pseudomonas syringae No. 31 suspensions, particularly at temperatures above — 5°C. The nucleation frequency of strain KUIN- 1-suspensions was similar to that obtained for an ice-nucleating Erwinia herbicola No. 26 at — 5°C.     

Studies on the relationship between the pathogenicity of Paracoccidioides brasiliensis in mice and its growth rate under different oxygen atmospheres

We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 °C. 10⁶ units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4. Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity.     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.     

Freezing Resistance and Cryopreservation of Cell Strains of Rhaponticum carthamoides and Thalictrum minus

Effects of long-term (few months) culturing and short pregrowth (up to 7 days prior to deep freezing) in the presence of mannitol (5–6%), ABA (5.0–7.5 × 10^–5 M), or both substances on cryogenic resistance of leusea (Rhaponticum carthamoides, strains Rhs-2 and Rhs-8) and meadow rue (Thalictrum minus L., strain B-233) cell suspension cultures were studied. Cryoprotective capacities of 48 solutions were studied at slow (0.33°C/min) freezing to the temperature of liquid nitrogen with an automatic initiation of crystallization. Cells were stored in liquid nitrogen for several days or months. ABA had a cryoprotective effect, provided that subculturing intervals were 12–14 days. At more frequent subculturing (every 7 days), pregrowth on ABA-containing medium did not increase survival percentage compared to pregrowth with mannitol. Successful cryopreservation of these strains has been achieved due to strict standardization of 7-day subculturing regime, pregrowth in the presence of mannitol prior to freezing, and cryopreservation with dimethyl sulfoxide, sucrose, trehalose, and glycerol. The cell survival rates after thawing were 60% (Rhs-8), 80% (Rhs-2), and 70% (B-233). The cell growth resumed on the third to seventh day. The growth indices and protoberberine synthesizing activity in B-233 strain reached their control values at the ninth subculturing after a post-thaw recovery.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION IN HYOSCYAMUS NIGER

Culture of segments of leaves, petioles, and stems and of germinated seeds and whole ovaries enclosing unpollinated ovules of Hyoscyamus niger in a medium containg 2, 4-dichlorophen-oxyacetic acid caused massive growth of calluses. In cultured germinated seeds and ovaries, calluses were formed from the hypocotyl and placenta, respectively. Leaf and stem explants generally gave rise to hard, white, and compact calluses. Upon transfer of these calluses to an auxin-free medium they regenerated roots or buds but showed no further signs of morphogenesis. Calluses formed on petioles, germinated seeds, and ovaries were a mixture of the white, compact and brown, friable types. Subsequent morphogenesis of the friable calluses in a medium lacking auxin was characterized by the formation of somatic embryos whose pattern of development was similar to that of zygotic embryos.     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

Improved growth conditions for hyphomicrobium sp. B-522 and two additional strains

Growth yields and rates of 3 hyphomicrobia were improved by varying components of or adding compounds to medium 337. Methanol (0.5% v/v), and similarly methylamine·HCl (3.38g/l), were optimal among 22 C-sources tested; increasing the methylamine·HCl concentration to 5.07g/l gave higher Hyphomicrobium B-522 yields but also prolonged lag periods. Ten C-sources (organic acids, alcohols) stimulated growth slightly but significantly, even in subcultures. Sugar compounds were not utilized. Strains B-522 and ZV-580 were stimulated by l-lysine and gluconate, while NQ-521 gr was stimulated by aspartate.N-Sources tested were inorganic (3), organic (3), or complex (3). (NH₄)₂SO₄ (0.5g/l) was optimal for strains ZV-580 and NQ-521 gr, but Hyphomicrobium B-522 grew best with urea-N. With NH ₄ ⁺ , strain B-522 grew as homogeneous suspension, all other N-sources caused clumping and pellicle formation. Inorganic requirements (PO ₄ ^3- , Mg, Ca, Fe, Mn, Mo) of strains B-522 and ZV-580 were optimized. Addition of Ni, Co, or Zn had no effect; metals 44 or Cu, resulted in growth inhibition.Vitamin B₁₂ stimulated Hyphomicrobium B-522; 2.5g/l B₁₂ decreased the doubling time from 9.3–10.8h to 5.4–5.8h. All combined single improvements resulted in a protein increase of 557% (B-522), 141% (NQ-521 gr), or 109% (ZV-580), respectively.     

Mannose accommodation of <Emphasis Type="Italic">Vigna angularis</Emphasis> cells on solid agar medium involves its possible conversion to sucrose mediated by enhanced phosphomannose isomerase activity

Mannose is an unusable carbon source for many plants. In our study we compared the effects of mannose and sucrose on growth and sucrose levels in azuki bean (Vigna angularis) cells grown in liquid media and in solid media. The suspension cells grew actively in a liquid medium containing 90 mM sucrose but not in that containing 90 mM mannose, where the intracellular sucrose levels were reduced to 20% or less of those in sucrose-grown cells. These results suggested that the limited conversion of mannose to sucrose resulted in cell growth inhibition. When sucrose-grown suspension cells (1 × 10⁵) were transferred onto agar medium containing mannose, they grew little initially, but, after a month lag period, they started to form many callus colonies at a high apparent variation rate (1.3 × 10⁻³). Time-course studies for sugar and enzyme analysis revealed that the mannose-accommodated cells were capable of converting mannose to sucrose, with enhanced phosphomannose isomerase activity. The mannose-accommodated cells actively grew in liquid medium with sucrose but lost their ability to grow with mannose again, suggesting a specific trait of callus culture for mannose utilization. The possible differences in the metabolic activities and other physiological characteristics are discussed between callus and suspension cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Thalassospira permensis</Emphasis> sp. nov., a new terrestrial halotolerant bacterium isolated from a naphthalene-utilizing microbial consortium

A halotolerant bacterium, strain SMB34^T, was isolated from a naphthalene-utilizing bacterial consortium obtained from primitive technogeneous soil (Verkhnekamsk salt deposit, Perm region, Russia) by enrichment procedure. The strain itself was unable to degrade naphthalene and grew at NaCl concentrations up to 11% (w/v). The 16S rRNA-based phylogenetic analysis showed that the strain belongs to the genus Thalassospira. The DNA-DNA hybridization values between SMB34^T and the type strains of phylogeneti-cally closest species (T. xiamenensis, T. profundimaris and T. tepidiphila) did not exceed 50%. The novel strain could be distinguished from the above species by the cell motility, MALDI/TOF mass spectra of whole cells and a range of physiological and biochemical characteristics. SMB34^T also considerably differs from the recently described species T. xianhensis, with the most striking differences in the DNA G + C content (53. ± 1.0 vs. 61.2 ± 1.0 mol %) and predominant ubiquinones (Q-10 vs. Q-9). The data obtained suggest strain SMB34^T (=VKM B-2527^T = NBRC 106175^T), designated as the type strain, represents a novel species, named Thalassospira permensis sp. nov.     

Cytopathogenicity of Naegleria fowled for Cultured Rat Neuroblastoma Cells1

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to ⁵¹Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44°C for 60 min or at 60°C for 30 min. Cytotoxicity was stable during storage at 4°C or at ?20°C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4°C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

Spontaneous mutants of Microsporon gypseum resistant to griseofulvin

Summary 1) The spores of the microconidial mutant I–18 of the dermatophyteMicrosporon gypseum in agar medium with GF germinated and formed germ tubes deformated in a characteristic way. From 1µg GF/ml up with an increasing antibiotic concentration (expressed in logarithms) the munber of colonies grown (expressed in probits) decreased linearly.2) As a sensitivity measure of the spores the median efficient dose ED 50 was used which was determined by means of a graphic probit analysis. For the strain used this value was determined in the range between 1.35–1.95µg GF/ml in three independent experiments.3) From the smears of a thickened spore suspension (1.6–14.2 × 10⁷ viable spores) in medium containing a high GF concentration a very small, but as for the order a stable number of colonies grew, as found in eight independent experiments. On the medium containing 20µg GF/ml in average 61 colonies grew, on 40µg GF/ml 20 colonies, on 80µg GF/ml 3 colonies and on 160µg GF/ml 0.3 colony (expressed in 10⁷ viable spores tested).4) A part of these colonies were isolated and transferred 29 times on a medium without the antibiotic. Two isolates only show a permanently increased resistance to GF, viz. the strain D-29 which is 50 × more resistant and the strain N-53 which is 3.5 × more resistant than the wild strain I-18.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri

Summary Production of vitamin B-12 compounds from methanol was carried out by Methanosarcina barkeri Fusaro, an anaerobic methanogen. The methanogen released about 40% to 70% of corrinoids irrespective of the culture medium used. The use of cysteine instead of Na₂S as the sole sulphur source for cell growth led to an increase in the cobalt chloride concentration in the culture medium up to 16 times the normal (0.6 mg·l^-1) without medium precipitation. This in turn resulted in an intracellular vitamin B-12 content of 5.6 mg·g dry cell^-1, the rest being discharged into the culture supernatant; this was 87 mg·l^-1, 73% of the total corrinoids after 20 repeated intermittently fed cultures and the final cell concentration was 5.8 g dry cell·l^-1. Taking advantage of this, continuous production of extracellular vitamin B-12 compounds was attempted with a fixed-bed bioreactor (carrier: diatomaceous clay). At a steady state operation at space velocity of 9 to 11 day^-1, the concentration of the discharged corrinoid was 6.8 to 7.9 mg·l^-1, having a vitamin B-12 activity of about 4 mg·l^-1. Total cell mass retained in the reactor was 39.6 g dry cell l-reactor^-1. Identification of the corrinoids revealed that 19% of the total corrinoids was comprised of the vitamin B-12 Factor III (5-hydroxybenzimidazolyl cobamide) and the remainder were mainly the base-free vitamin B-12 Factor B (cobinamide and its derivatives).     

Growth of <Emphasis Type="Italic">E. coli</Emphasis> BL21 in minimal media with different gluconeogenic carbon sources and salt contents

Escherichia coli strain BL21 is commonly used as a host strain for protein expression and purification. For structural analysis, proteins are frequently isotopically labeled with deuterium (²H), ¹³C, or ¹⁵N by growing E. coli cultures in a medium containing the appropriate isotope. When large quantities of fully deuterated proteins are required, E. coli is often grown in minimal media with deuterated succinate or acetate as the carbon source because these are less expensive. Despite the widespread use of BL21, we found no data on the effect of different minimal media and carbon sources on BL21 growth. In this study, we assessed the growth behavior of E. coli BL21 in minimal media with different gluconeogenic carbon sources. Though BL21 grew reasonably well on glycerol and pyruvate, it had a prolonged lag-phase on succinate (20 h), acetate (10 h), and fumarate (20 h), attributed to the physiological adaptation of E. coli cells. Wild-type strain NCM3722 (K12) grew well on all the substrates. We also examined the growth of E. coli BL21 in minimal media that differed in their salt composition but not in their source of carbon. The commonly used M9 medium did not support the optimum growth of E. coli BL21 in minimal medium. The addition of ferrous sulphate to M9 medium (otherwise lacking it) increased the growth rate of E. coli cultures and significantly increased their cell density in the stationary phase. An erratum to this article can be found at     

Fontivita pretiosa gen. nov., sp. nov., a thermophilic planctomycete of the order Tepidisphaerales from a hot spring of Baikal lake region

A novel facultatively anaerobic moderately thermophilic bacterium, strain B-254^T, was isolated from a terrestrial hot spring near the town of Goryachinsk in the Baikal lake region (Russian Federation). Motile spherical cells of the strain were present as single cocci, in pairs, or aggregates. The cells had a Gram negative cell wall and reproduced by binary fission. The isolate grew at 30–57 °C (opt. 50–54 °C) and at pH 5.1–8.4 (opt. 6.6–7.1). Strain B-254^T was a chemoorganoheterotroph, growing on mono-, di- and polysaccharides (xylan, starch, galactan, galactomannan, xyloglucan, arabinan, curdlan, beta-glucan, locust bean gum, xanthan gum). Sodium chloride or yeast extract were not required for growth. Major cellular fatty acids were iso-C_16:0, anteiso-C_17:0, and C_20:0; major polar lipid was phosphatidylethanolamine. The complete genome of strain B-254^T was 5.54 Mb; its GC content was 64 %. According to the results of 16S rRNA gene sequence-based phylogenetic analysis and the conserved proteins sequences-based phylogenomic analysis strain B-254^T was on a separate lineage within the order Tepidisphaerales (Phycisphaerae, Planctomycetes). Based on phylogenetic and phylogenomic analyses of Phycisphaerae, whole genome comparisons of Tepidisphaerales as well as distinctive phenotypic features of the strain, it was assigned to a novel genus and species for which the name Fontivita pretiosa gen. nov. sp. nov. is proposed. Strain B-254^T = KCTC 82380^T = VKM B-3507^T.     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Morphogenesis in callus tissue ofMedicago sativa: The role of ammonium ion in somatic embryogenesis

Exogenously supplied ammonium ion is critical to alfalfa morphogenesis in vitro. In alfalfa, the ability to induce the formation of either roots or somatic embryos provided an opportunity to examine the effects of ammonium ion on each pattern of morphogenesis. Somatic embryo formation required a minimum of 12.5 mM NH ₄ ⁺ in regeneration medium for optimal expression. Root formation was inhibited by NH ₄ ⁺ levels of 50 mM and above, and occurred in the absence of exogenous NH₄ ⁺. At high levels of NH ₄ ⁺ somatic embryos were formed from cells exposed to the root-inducing combination of hormones. This observation suggests that the growth regulators and exogenously supplied ammonium ion comprise an interactive system controlling the in vitro pattern of alfalfa morphogenesis.