Growth Behavior of a Liverwort, Jungermannia subulata Evans, in a Cell Suspension Culture. The Role of Organic Acids Required for Cell Growth

A green callus of a liverwort, Jungermannia subulata Evans,was induced from gametophytes, and a cell suspension culturewas obtained from the callus. Both callus and suspension-culturedcells grew in a modified Murashige and Skoog medium if organicacids of the TCA cycle were supplied. In the cell suspensionculture of J. subulata, ammonium was taken up preferentiallyby the cells particularly at the earliest stage of growth, whileonly a negligible amount of nitrate was utilized as long asammonium was present in the medium, and this unbalanced utilizationof the two nitrogen sources caused an abrupt drop in the pHof the medium. The organic acids of the TCA cycle supportedthe growth of this cell line by preventing the abrupt drop inthe pH of the medium. (Received June 13, 1981; Accepted October 8, 1981)     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

Improved Growth of Tissue Cultures of the Onion, Allium cepa

A forty-fold increase in tissue fresh weight has been achieved for Allium cepa stem tissue callus grown on a modified B5 medium with increased ammonium, phosphate and nitrogen levels; and decreased level of 2,4-dichlorophenoxyacetic acid. Growth was examined on several standard media, using fresh weight, dry weight and cell number as parameters over an 8-week growth period. Further, tissue grown upon this medium is more friable than on other media. The value of such friable callus for suspension culture initiation and for single cell cloning experiments is discussed.     

Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by Silene vulgaris cell culture

Medium nutrients such as calcium, phosphorus, nitrogen and nitrate to ammonium ratio have significant influence on the growth, biosynthetic and biochemical characteristics of polysaccharides produced by Silene vulgaris (M.) G. cell culture. Cell growth and production of polysaccharides was limited by an absence of any of these components in the medium. Optimal growth of the callus and production of arabinogalactan were achieved at 1.5-4.5 microM calcium while the optimal production of pectin named silenan was observed at 3.0-4.5 microM. The phosphate contents in the medium in the range of 0.63-3.75 microM were favorable for callus growth. Production of silenan was maximal at 1.25-3.75 microM phosphate. Optimal growth of the callus was achieved at 30-90 microM nitrogen. Maximal production of silenan was observed at 60 microM nitrogen while the optimal production of arabinogalactan was at 90 microM nitrogen (at ratio of NH(4)(+):NO(3)(-) as 1:2). A presence both of nitrate and ammonium is needed for the silenan biosynthesis (the NH(4)(+):NO(3)(-) ratio as 1:1 and 1:2). Yields and volumetric production of arabinogalactan were maximal at deletion of ammonium from the nutrient medium (ratio 0:1). Absence of calcium or nitrogen in the medium leads to a decrease of the galacturonic acid residues in silenan. The galactose residues contents in arabinogalactan were decreased in the absence of nitrogen and calcium in the medium.     

Effect of different nitrogen sources on the biosynthesis of lipase by Oospora lactis

The effect of inorganic and organic nitrogen compounds on the synthesis of biomass and extracellular lipase by Oospora lactis was studied. Among the inorganic nitrogen sources ammonium sulphate and ammonium secondary phosphate and among the organic nitrogen sources yeast autolysate proved to be most beneficial for the lipase synthesis. Lipase activity and biomass accumulation in the medium containing yeast autolysate were greater than in the media containing the above ammonium salts. Lipase synthesis reached maximum in the nutrient medium containing yeast autolysate (0.7%) and ammonium sulphate (0.3%).     

NUTRITIONAL REQUIREMENTS FOR OVULE FORMATION IN EXCISED PISTILS OF NIGELLA

The nutritional requirements for ovule formation in Nigella saliva L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mil) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mM nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of α-alanine (1 mM) and γ-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high nitrate medium.     

Control of pH and its Effect on Ammonium Utilization by Nitrate Reductase Deficient Plants,Cells, and Protoplasts of Nicotiana tabacum

Nitrate reductase deficient plants of Nicotiana tabacum were unable to utilize ammonium efficiently unless the medium was buffered against excessive acidity accumulation. In addition to succinate, calcium carbonate, MES and phosphate buffers allowed plants to utilize ammonium efficiently. Similar observations were made regarding callus derived from stem tissue of these plants. Plants could be grown in compost, in a physiological state suitable for protoplast isolation, when watered with nutrient solutions containing NH₄NO₃ and MES buffer. Protoplast division and proliferation to the stage of plant regeneration was possible using Murashige and Skoog (1962) basal medium buffered with succinate, calcium carbonate or MES but not phosphate.     

Initiation, Growth and Nuclear Characteristics of Tissue Cultures of Paeonia suffruticosa

Tissue cultures of the garden paeony, Paeonia suffruticosa have been established using explants of etiolated stems. Callus formation was induced on agar-solidified media containing ammonium ions or amino acids together with the hormones 2,4-dichlorophenoxyacetic acid and kinetin, but not on media lacking the reduced nitrogen component. Attempts to induce callus from explants from green plants were completely unsuccessful and were characterized by the production of intense brown colorations, both of the explant and the medium. Subcultured tissue without the added hormones produced roots, both on solid and liquid media. Growth was tested on a range of liquid media, SH/2, SH, SH × 2 and SH—M, containing 1250, 2500, 5000 and 2500 mg/l potassium nitrate. The SH—M medium also contained 1650 mg/l ammonium nitrate. Growth measured as increased fresh weight was best in the SH/2, SH and SH—M media and was curtailed in the SH × 2 medium. Soluble protein content was highest at the lowest nitrogen concentration. A histochemical comparison of tissue grown on the SH/2, SH—M and SH—M lacking hormones showed that the cells in all the cultures remained diploid while differences were established in total nuclear protein, measured using the ninhydrin-Schiff procedure. Nuclei from SH—M grown cells have a higher protein content than those from the SH/2 medium while cells from the SH—M medium lacking hormones show a further increase in nuclear protein. This raises the question whether this change in nuclear protein is related to the morphogenesis of roots which occurs in this medium.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

Studies on aspergilli XVII. Effect of nitrogen sources on growth and fruiting

Summary The effect of ammonium, nitrate, and organic nitrogen on growth and sporulation of 18 Aspergilli was examined in a chemically defined medium in surface culture under controlled conditions. All three forms of nitrogen were metabolized by all the Aspergilli tested. Ammonium nitrogen was not good both for growth and fruiting. This was due to the sharp fall in the pH level which resulted due to the rapid utilization of anions of the ammonium nitrogen than cations. The effect of adding succinic acid in the medium containing ammonium nitrogen has been discussed.Good growth of Aspergilli in media containing nitrate nitrogen with the accompanying rise in the pH of the medium showed that these species are capable of reducing nitrate nitrogen to the level of ammonia. The role of succinic acid in the utilization of nitrate nitrogen was investigated. All fungi accomplished good growth on a medium containing asparagine.     

The Cultivation of Tissues of Cupressus lusitanica in vitro

Conditions for In vitro culture of initial explants of Cupressus lusitanica were determined as the first step of an investigation of physiological differences between tissues obtained from plants, or plant parts, of different ages. Explants proliferated well on a basal agar medium containing only Heller's mineral salts and sucrose. Addition of several vitamins, phytohormones, and coconut water to this basal medium stimulated callus growth only slightly or not at all, and transplanted callus soon died. Proliferation of initial explants was considerably stimulated by increasing the concentration of mineral salts in the medium, and the growth habit changed from compact to friable. Callus could be transplanted to, and maintained on, a high-salt medium enriched by vitamins. Whereas added auxins still stimulated proliferation of initial explants and transplanted callus only slightly, added coconut water was considerably more effective than on basal medium. Thus results confirm observations by others with tobacco tissues: Additions of phytohormones and other organic substances may not exert their potential effects if growth is limited by sub-optimal mineral nutrition.     

Effect of nitrogen sources on cellulase biosynthesis by a mutant strain of Trichoderma viride 44

The effect of various nitrogen sources on cellulase biosynthesis by the mutant strain Trichoderma viride 44 was examined. This strain may utilized nitrogen in the nitrate, ammonium of organic form. When cultivating this strain, it appears advantageous to add to the nutrient medium yeast and yeast lyzates as well as their mixture with ammonium sulfate. Cellulase reached its maximum activity of 20.2, 21.5 and 23.2 mu/ml when grown on the medium containing ammonium phosphate, peptone and brewing yeast plus ammonium sulfate, respectively. It is useful to apply nitrogen in its organic forms in small quantities and in combination with mineral forms. The nitrogen presence in the medium is necessary only at the exponential stage of fungal growth. The lack of nitrogen in the stationary stage characterized by the maximum cellulase formation does not inhibit an increase in the enzyme activity.     

Influence of auxins,cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (<Emphasis Type="Italic">Morus</Emphasis><Emphasis Type="Italic">alba</Emphasis> L.)

Rutin is an economically valuable flavone compound with anticancer activity, dietary effects, and anti-aging activity. In this study, callus and adventitious roots were induced from three Morus (mulberry) species. Among the three mulberry species tested for rutin production, roots of the Sugye (M. alba L.) had the highest levels (242.2 μg/g fresh tissue) of rutin. In addition, the mature leaves of this type of tree promoted higher levels of rutin compared to those of young leaves or those undergoing senescence. Adding auxins such as indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) not only enhanced the development of callus and adventitious roots but also increased the protein and rutin contents. In contrast, adding cytokinins such as 6-benzyladenine (BA) and kinetin (KN) retarded callus and adventitious root development as well as the protein and rutin contents. Callus in suspension culture in the presence of IAA produced more rutin than that in the absence of IAA. However, rutin secretion into a medium was greater in the absence of IAA. Different ammonium/nitrate (AM/NI) ratios in a root suspension culture also greatly affected rutin production and its secretion into a liquid medium. As a result, the highest level of rutin was produced when adventitious roots were grown in a 34/66 AM/NI full-strength standard MS medium containing 5 mg/l IAA.     

THE HORMONAL CONTROL OF ORGAN FORMATION IN CALLUS OF MEDICAGO SATIVA L. CULTURED IN VITRO

Organogenesis in alfalfa callus (Medicago sativa L. cv. ‘Regen S’) has been obtained by the transfer of callus from an induction medium containing growth regulators to a regeneration medium lacking growth regulators. The transfer of callus from induction medium containing high levels of 2,4-D and low levels of kinetin to regeneration medium resulted in the formation of shoots. Conversely, the transfer of callus from induction medium containing low levels of 2,4-D and high levels of kinetin resulted in the formation of roots. The pattern of organogenesis on regeneration medium was modified by the nutritional composition of that medium. When Blaydes medium supplemented with inositol and yeast extract was employed as regeneration medium, root organogenesis was inhibited. Root organogenesis was not inhibited by either Shenk and Hildebrandt medium or Gamborg's B5 medium. Shoot formation occurred on all of these media. A survey of the in vitro organ-forming capacity of 14 genotypic clones from the cv. ‘Regen S’ was conducted. The capacity to form organs differed quantitatively among the clones analyzed. A more detailed analysis of a highly responsive clone (RA3) and a poorly responsive clone (RA5) revealed no significant qualitative difference in their organogenic responses.     

Morphogenesis in lentil: Plant regeneration from callus cultures of Lens culinaris medik. Via somatic embryogenesis

Plants were regenerated by the process of somatic embryogenesis from embryo-derived callus cultures of Lens culinaris Medik. cv. Laird. The callus originated from embryonal axes cultured on Murashige and Skoog (MS) (Physiol. Plant., 15 (1962) 473) medium or on a modified B5 (Gamborg et al. Exp. Cell Res., 50 (1968) 151) medium (containing 500 mg · 1⁻¹ ammonium nitrate, designated B5A) supplemented with 1–10 mg · 1⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was pale white, friable, organized and slow growing. On transfer to B5A medium without hormones or with benzyladenine (BA) and indoleacetic acid (IAA), certain peripheral areas of the callus turned green. Such green patches later differentiated into several embryoid-like structures. Further subculture of the embryoid-like structures (or embryoids) on a glutathione-supplemented medium produced well organized embryos having cotyledons, shoots and roots which were able to develop into whole plants.     

NaCl-Dependent growth, ion content and regeneration of calluses initiated from the mangrove plant,Bruguiera sexangula

Calluses initiated from leaves and seedlings of the mangrove,Bruguiera sexangula, were isolated from the original tissues and subcultured. Effects of NaCl on growth and ion content of each callus were measured. The growth rate of calluses derived from leaves (leaf callus) gradually decreased as the NaCl concentration in the medium increased, while that of calluses derived from seedlings (seedling callus) was highest in the medium containing 100 mM NaCl. Concentrations of Na and Cl in both calluses increased with increasing the NaCl concentration in the culture medium. The concentration of K of leaf calluses greatly decreased at 300 mM NaCl, while the K concentration of seedling calluses decreased only slightly and remained relatively high even in the presence of 300 mM NaCl. Transient treatment of leaf calluses with media containing high concentrations of NaCl frequently induced regeneration of adventitious tissues.     

Inorganic nitrogen source for autotrophic growth of Euglena mutabilis Schmitz

The effect of inorganic nitrogen source on population growth of Euglena mutabilis, an acidophillic benthic protozoa colonizing on the sediment of acid mine drainage, was investigated. Sodium nitrate, ammonium chloride, and ammonium sulfate were tested as nitrogen sources. The population density of E. mutabilis at equilibrium density cultivated in ammonium chloride‐ and ammonium sulfate‐containing media was 9–11 times higher than that in sodium nitrate‐containing medium at the optimal salt medium concentration. The population growth of E. mutabilis in ammonium sulfate‐containing medium was rapid and reached half of the equilibrium density after ca. 228 h, which was ca. 77 h earlier than that in ammonium chloride‐containing medium. Culture medium with ammonium sulfate as the nitrogen source achieved the highest maximum population density and the fastest growth rate among the three nitrogen salts used as nitrogen sources.     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

Indirect somatic embryogenesis of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L<Emphasis Type="Italic">.</Emphasis> in liquid medium and improvement of embryo-to-plantlet conversion rate

The establishment of cocoa embryogenic cell lines in liquid medium starting from high frequency somatic embryogenesis (HFSE) callus is described. The growth kinetics of the cultures during the multiplication and the expression steps conducted in 250 mL Erlenmeyer flasks were described for three genotypes selected for their agronomical traits (EET95, EET96, and EET103). The glucose and dissolved oxygen concentrations and the absorption of Murashige and Skoog medium macronutrients (nitrate, ammonium, potassium, sulfate, calcium, phosphorus, and magnesium) were monitored. The multiplication of the embryogenic calluses in a medium containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 mg L^?1, initiated with an inoculation density of 20 g L^?1 of callus, was achieved. The growth rate was characterized by two phases, with the second being concomitant with a depletion of phosphorus and magnesium, and a decrease in the embryogenic potential of the callus. The expression of the callus embryogenic capacity was conducted in an auxin-free medium. The embryo production starting from 1 and 5 g L^?1 inoculation densities was compared. When placed in the optimal expression conditions in flasks, 1 g of callus produced 1000 to 1500 embryos within 5 to 7 wk. Finally, two paths for improving the plantlet regenerative capacities of cocoa SE produced in liquid medium were identified. Supplementing the expression medium with myo-inositol used as an osmotic agent at a concentration of 50 g L^?1 increased the embryo-to-plantlet conversion rate from 13–16% to 40–48%. A 6-wk culture of the embryos on a maturation medium in Petri dishes optimized their subsequent development into plantlets.     

Effects of ammonium, L-glutamate, and L-glutamine on nitrogen catabolism in Aspergillus nidulans

During growth of Aspergillus nidulans in medium containing ammonium the specific activities of most enzymes involved in catabolism of nitrogen sources are low (ammonium repression). The gdhA10 lesion, which results in loss of nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase activity, has been shown to lead to partial relief of ammonium repression of three amidase enzymes as well as histidase. The areA102 lesion led to altered levels of these enzymes but did not greatly affect ammonium repression. The double mutant areA102,gdhA10 was almost completely insensitive to ammonium repression of two of the amidase enzymes and histidase. This suggests that an interaction between the areA and gdhA genes in determining responses to ammonium occurs. Growth of mycelium in medium containing l-glutamate has been found to result in lowered levels of all four enzymes, and this occurs in strains insensitive to ammonium repression. Very strong repression in all strains occurred during growth in medium containing l-glutamine. Relief of these repressive effects of glutamate and glutamine was blocked by cycloheximide. Glutamate and glutamine had similar effects on the production of extracellular protease activity, and growth on glutamine led to low levels of urate oxidase. In contrast to the above enzymes, nitrate reductase was insensitive to the effects of glutamine and glutamate, even though this enzyme is very sensitive to ammonium repression. Although other possibilities exist, it is suggested that there may be mechanisms of general control of nitrogen-catabolic enzymes other than ammonium repression.