Ethylene Production by Tobacco (Nicotiana tabacum) Callus

Tobacco callus cultures grown on defined agar-solidified media produced ethylene in differing amounts, which were related to cultural treatment and age of the callus. There was a close correlation between the rate of ethylene production and growth. In darkness, maximal rates occurred in the third week of growth with ethylene production in the range of 750 nl (callus piece)^?1 d^?1 (fr. wt. = 1.5 g), and in the light, maximal rates occurred in the first week of growth, 200 nl (callus piece)^?1 d^?1 (fr. wt. = 200 mg). Growth was also correlated with ethylene production when the latter was altered by exposure of the callus to inhibitors of ethylene synthesis, L-canaline, benzyl isothiocyanate, and 3,5-diiodo-4-hydroxy-benzoic acid. No correlation was found following treatment with AgNO₃, a presumptive inhibitor of ethylene action. The inhibition of growth and ethylene production by L-canaline was partially reversed by gassing the cultures with ethylene (1 μl/1). A mercuric perchlorate sink had no significant effect on growth. A possible relationship between ethylene evolution and growth is discussed.     

Rice callus physiology – Identification of volatile emissions and their effects on culture growth

Rice ( Oryza sativa L.) callus cultures modified the atmosphere of the headspace of the vessel used for culture maintenance by producing carbon dioxide, ethanol and ethylene, while utilizing oxygen. Small quantities of acetaldehyde and ethane were also measured in one cultivar. Under these conditions of culture maintenance, callus from some cultivars were much more difficult to keep alive than callus from others. The largest proportion of necrotic callus was produced from cultures growing under conditions of high ethylene production and low oxygen utilization. When callus was exposed to a controlled flowing gas mixture similar to that accumulated above necrotic callus, growth of 2 necrosis-susceptible cultivars was more strongly inhibited than growth of 2 necrosis-tolerant cultivars. The poor callus growth was associated with higher ethylene production and a lower rate of oxygen utilization.     

Effects of ethylene on somatic embryogenesis and galanthamine content in <Emphasis Type="Italic">Leucojum aestivum</Emphasis> L. cultures

The influence of ethylene on in vitro morphogenesis of Leucojum aestivum and galanthamine accumulation was studied. Calli were cultivated on Murashige and Skoog (MS) medium supplemented with 25 μM 4-amino-3,5,6-trichloropicolinic acid (picloram) and 0.5 μM benzyladenine (BA). During incubation under these conditions, callus cultures produced ethylene (9.5 nL/g fresh weight: F.W.) whereas no ethylene was found in somatic embryos cultivated on medium supplemented with 0.5 μM α-naphthalene acetic acid (NAA) and 5 μM zeatin. Application of the precursor of ethylene 1-aminocyclopropane-1-carboxylic acid (ACC) increased ethylene production in both cultures, and decreased callus growth by a factor of 1.2, whereas callus growth was enhanced by a factor of 1.1 in the presence of an inhibitor of ethylene silver nitrate (AgNO₃) or by a factor of 1.2 with an absorbent potassium permanganate (KMnO₄). ACC enhanced the induction of somatic embryos and the development of globular embryos. Removal of ethylene by KMnO₄ during somatic embryogenesis led to the development of plants with greater length. Silver thiosulphate (STS) induced galanthamine production in callus cultures (0.1% dry weight), whereas ACC induced galanthamine production in somatic embryo cultures (2% dry weight).     

Endogenous ethylene in indirect somatic embryogenesis of <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Ethylene biosynthesis during different phases of somatic embryogenesis in Medicago sativa L. cv. Rangelander using two regeneration protocols, RPI and RPII, was studied. The highest ethylene production was detected during callus growth on induction medium in both regeneration protocols. Significantly less ethylene was produced by embryogenic suspension than by callus (RPII). Developing embryos synthesized higher amounts of ethylene than mature embryos. Production of ethylene was strongly limited by the availability of 1-aminocyclopropane-1-carboxylic acid and also by ACC-oxidase activity. However, removal of ethylene from culture vessels’ atmosphere using KMnO₄ or HgClO₄ had no significant effect on callus growth, somatic embryo induction and development. Reducing of ethylene biosynthesis by aminoethoxyvinylglycine substantially decreased somatic embryo production and adversely affected their development, indicating ethylene requirement during proliferation and differentiation but not induction.     

Ethylene production and toxigenicity of methionine and its derivatives with riboflavin in cultures of Verticillium, Fusarium and Colletotrichum species exposed to light

Ethylene was produced by Verticillium dahliae Kleb. grown in liquid Czapek's medium. The rate of ethylene production was enhanced by light but was not affected by shaking or the growth rate of the cultures. L-, D- and DL-methionine, DL-ethionine and a -keto- y -methylthiobutyric acid (KMBA) were good substrates for ethylene production. KMBA may be an intermediate in ethylene production and it appears to be degraded to ethylene either enzymatically by peroxidase or photochemically in the presence of riboflavin. Addition of riboflavin or briefly heating the cultures to 100°C enhanced ethylene production greatly, while the addition of sodium azide, potassium cyanide and catalase were very inhibitory. The SS4 (non-defoliating) pathotype of V. dahliae produced significantly more ethylene (up to 108.4 nl ethylene h₁ from 20 ml-10-day-old cultures) than did the T9 (defoliating) pathotype with all substrates tested. The results suggest that the in vitro rate of ethylene production is not related to the relative virulence of pathotypes of V. dahliae on cotton. A number of Verticillium species, Fusarium oxysporum f. sp. vasinfectum and Colletotrichum dematium var. truncatum were able to produce ethylene in liquid Czapek's medium containing 1 m M L-methionine under continuous light. Riboflavin, although highly stimulatory to ethylene production, caused a fungicidal reaction to all the fungi tested in Czapek's medium containing L-methionine under continuous light. The fungicidal effect of the riboflavin-methionine-light combination occurred at concentrations of riboflavin and methionine less than 1.33 μ M and 0.5 m M , respectively. No fungicidal activity was detected when the cultures were grown in total darkness or when either methionine or riboflavin was omitted from the culture medium.     

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m^?2 s^?1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m^?2 s^?1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.     

Light-susceptibility of camptothecin production from<Emphasis Type="Italic">in vitro</Emphasis> cultures of<Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne

Production of camptothecin (CPT) from callus cultures ofCamptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light 90∶10.     

Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures

Carrot cells were cultured under various light spectra and intensities at different times following the initiation of suspension cultures from callus. The highest intensity white and blue light treatments were inhibitory to growth and somatic embryogenesis. Red and green light were not different from dark treatments which produced the highest total number of embryoids. After extended time in culture, carrot cells in blue light produced secondary embryoids and anthocyanin. Cultures in red light had multiple cotyledons and orange-pigmented radicles. Leafy cotyledons occurred in all light treatments. Abscisic acid production peaked at the heart stage of embryogenesis and synthesis was most pronounced in blue light. Red light enhanced development to the heart stage. Both the red and blue light spectra may be used to manipulate carrot cell cultures to optimize growth.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Endogenous Gibberellins and Growth of Tobacco Callus Cultures

Tobacco callus grown under a range of conditions for different lengths of time contained various levels of gibberellin-like substances. Culture conditions, viz: light versus darkness and the quantity of cytokinin in the medium, affected the amount of gibberellins found in the tissue. These culture conditions were also important in controlling growth rate of the callus and modified the ability of the tissue to respond to exogenous gibberellins. Furthermore, substances which are known to inhibit gibberellin biosynthesis and also thought to block gibberellin action in some cases, were found to reduce the rate of growth. These data support the idea that endogenous gibberellins may be important in the control of the normal growth of tobacco cells in culture.     

Induction of a photomixotrophic plant cell culture of Helianthus annuus and optimization of culture conditions for improved α-tocopherol production

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l^?1 sucrose, 0.5 mg l^?1 of the auxin 1-naphthalene acetic acid, and 0.5 mg l^?1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.     

Defined conditions for the initiation on growth of cotton callus in vitro I.Gossypium arboreum

Summary Defined in vitro conditions for callus initiation byGossypium arboreum L. were determined, and different tissues were evaluated as explant sources. Environmental conditions tested included light versus dark, and low light versus high light. Different nutrient media as well as carbohydrate sources were examined. Our data show that hypocotyl tissue was superior to cotyledon or leaf tissue as the explant source for callus proliferation; the Murashige-Skoog inorganic formulation with (in mg per 1) 100 myo-inositol, 0.4 thiamine·HCl, 2 indoleacetic acid (IAA), 1 kinetin, and 3% glucose solidified by agar was the best medium to initiate callus. Cultures with sucrose as a carbohydrate source browned rapidly. Callus proliferation was superior under high light (8000 to 9000 lux) conditions at 29±1°C. Various combinations of auxins and cytokinins were tested for their ability to improve callus proliferation and subsequent growth of subcultures. Although the MS medium containing IAA and kinetin was found superior for obtaining rapid proliferation of callus from hypocotyl explants, a second medium containing 2 mg per 1 naphthalenacetic acid (NAA) and 0.5 to 1 mg per 1 benzyladenine (BA) was found necessary for vigorous growth of subcultured callus. A MS medium with 5 to 10 mg per 1 {ie329-1} (2iP) and 1 mg per 1 NAA was also favorable for continued subculturing. Technical Article 12485 from the Texas Agricultural Experiment Station.     

Enhancement of production and regeneration of embryogenic type II callus in Zea mays L. by AgNO3

In order to evaluate the impact of ethylene in maize tissue culture, silver nitrate has been used as an inhibitor of ethylene action. Type II callus initiation rate was improved when immature embryos were cultured on a modified Murashige & Skoog medium containing various concentrations of silver nitrate (5, 10, 20 mgl^-1). Regeneration ability of calli initiated and maintained in presence of silver nitrate was enhanced. No modification of callus growth rate neither of ethylene production has been detected.     

Studies on lavandin callus cultures: Ethylene production in relation to the growth

Ethylene production and growth of callus cultures of lavandin (Lavandula offidnalis Cham x Lavandula latifolia Villars) cv. Grosso were examined. Callus lines, derived from various kinds of primary expiants (shoot tip, leaf and calyx), exhibited differences in ethylene production patterns independent of callus growth. Moreover these differences could not be ascribed to the expiant source. Within a line, ethylene pattern paralleled callus growth curve. Variations in ethylene evolution were induced in shoot tip callus by means of ACC, AVG and varied amounts of 2,4-D in the culture medium. Following all these treatments callus growth was not altered. Hie decrease in 2,4-D concentration caused changes in Chl a and water content of the tissues.     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.     

Requirement of Ethylene for Growth of Callus and Somatic Embryogenesis in Medicago sativa L.

The role of ethylene in the growth of callus and somatic embryogenesisin Medicago sativa was examined. The application of 2,5-norbornadiene,a competitive inhibitor of ethylene action, during a 10 d inductionperiod to medium containing 2,4-D and kinetin inhibited thegrowth of callus but did not affect somatic embryogenesis, nordid it affect ethylene production during the induction stage.The exposure of tissue, incubated on differentiation medium,without hormones, to an atmosphere of 2,5-norbornadiene, inhibitedboth growth and embryo maturation and stimulated pigmentation.The inhibition of embryo maturation was observed even in thepresence of norbornadiene at a concentration which did not affectgrowth of tissue. It is suggested that the action of endogenous ethylene is necessaryfor the growth of the callus and embryo maturation. Key words: Medicago sativa, ethylene, callus growth, somatic embryogenesis     

Effects of growth condition on the structure of glycogen produced in cyanobacterium Synechocystis sp. PCC6803

Growth and glycogen production were characterized for Synechocystis sp. strain PCC6803 grown under continuous fluorescent light in four variations of BG-11 medium: either with (G+) or without (G−) 5 mM glucose, and with a normal (N+, 1.5 g sodium nitrate/L) or a reduced (N−, 0.084 g sodium nitrate/L) nitrogen concentration. Glucose-supplemented BG-11 with a normal nitrogen concentration (N+G+) produced the highest growth rate and the greatest cell density. Although the maximum cell mass production was observed in the N+G+ medium, the highest glycogen yield (19.0 mg/g wet cell mass) was achieved under the glucose-supplemented, nitrogen-limiting condition (N−G+). The addition of glucose enhanced cell growth, while nitrogen limitation apparently directed carbon flux into glycogen accumulation rather than cell growth. Transmission electron microscopic analysis showed that, under nitrogen-limiting conditions (N−G+), glycogen particles accumulated in large amounts and filled the cytosol of the cells. Analysis by high-performance size-exclusion chromatography further revealed that the glycogen produced in N−G+ medium had the longest average branch chain-length (DP10.4) among the conditions tested. When the yield and structure of glycogen were examined in different growth phases, the greatest yield (36.6 mg/g wet cell mass) and the longest branch chain-length (DP10.7) were observed 2 days after the fully grown cells in the N+G+ medium were transferred to the growth restricting (N−G+) medium.     

Rosmarinic acid synthesis in transformed callus culture of Coleus blumei benth

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.     

Rapid production of wheat cell suspension cultures directly from immature embryos

The aim of this study was to produce suspension cultures of winter wheat directly from immature embryos bypassing the callus stage, and to determine their capacity for growth and regeneration in comparison to suspension cultures produced from callus. The study was carried out using Polish winter wheat varieties: ‘Grana’ and ‘Rosa’. Immature embryos were isolated, homogenized and transferred directly to liquid medium supplemented with 2,4-D. Actively dividing cell cultures were obtained within 2 months after the cultures were started. Suspension cultures from callus of immature embryos was also produced. With both cultivars, faster growth was observed in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Metabolic activity was higher in the suspension culture produced directly from embryos than in the suspension derived from callus only in ‘Grana’. The production of 1-amiocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, was lower in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Morphogenic capacity was significantly higher in aggregates derived directly from embryos than in aggregates derived from callus. With ‘Rosa’, about one third of the aggregates derived directly from embryos regenerated shoots. Production of ACC was lower in ‘Rosa’ cell culture that regenerated then in other cell cultures that did not. Photosystem II reactions were more efficient in dark green aggregates than in light green or pale green aggregates which were unable to regenerate. With the method presented, wheat cell suspension cultures with a regeneration potential can be produced in 2 or 3 months less time than with traditional methods.     

Ethylene and the Responses to Light of Rice Seedlings

The growth of rice seedlings (Oryza satira L.) in the presence of ethylene caused a change in the response to light of coleoptile elongation. In plants grown in air without added ethylene coleoptile elongation was promoted by red, far-red and yellow-green light only in very young seedlings; in older plants irradiation inhibited the growth of the coleoptile. The effect of growing plants in the presence of ethylene was to prolong the period during which light promoted coleoptile growth. Elongation of the first internode was inhibited by light whether or not the seedlings were grown in the presence of ethylene. A correlation existed between the growth effect of an irradiation and the initial decay rate of phytochrome which was established by the treatment. Regardless of wave length, light sources whose intensities were adjusted to produce a decay rate of about 10% per hour or less induced a moderate rate of coleoptile elongation which persisted for a relatively long period. Irradiation with red or yellow-green light of higher intensity which produced a higher rate of phytochrome decay induced a higher rate of coleoptile elongation, but growth stopped after several hours. Other observations, however, showed that one cannot establish a general simple correlation between the rate of elongation of rice coleoptiles under light and the status of measurable phytochrome in the plant.