Ethylene Production by Plant Cell Cultures: Variations in Production during Growing Cycle and in Different Plant Species

Suspension cultures of Rosa sp., soybean (Glycine max L.), wheat (Triticum monococcum L.), sweet clover (Melilotus alba Desc.), Haplopappus gracilis Nutt., and rue (Ruta graveolens) produced ethylene. The amount varied with the species. The rate of formation in rose and Haplopappus cells paralleled growth but accelerated when the stationary phase was reached, after which the rate declined sharply. Light was not required for ethylene production. Exogenous ethylene could not replace 2,4-dichlorophenoxyacetic acid or naphthalineacetic acid in the cell cultures, and there was no stimulation of growth in the normal medium. Ethylene at 20 mm reduced growth of Ruta and rose cells by 30 and 20%, respectively. The amounts of ethylene produced by the cultures do not affect growth.     

Effect of silver nitrate on sugarcane cell suspension growth, protoplast isolation, ethylene production and shoot regeneration from cell suspension cultures

Silver nitrate (AgNO₃), an inhibitor of the physiological actionof ethylene, reduced cell growth, promoted ethylene production,increased the yield of protoplasts and reduced shoot regenerationfrom sugarcane heterogeneous cell suspension cultures. The increasein the rate of protoplast isolation from cultures treated withAgNO₃ (0 to 59 µM) correlate with an increase in endogenousethylene production by the cells. The addition to the culturemedium of chemicals that either inhibited (aminoethoxyvinylglycine,AVG) or promoted (aminocyclopropane-1-carboxylic acid, ACC)ethylene biosynthesis did not alter the number of protoplastsisolated from these cultures. However, protoplasts were isolatedwith AVG in combination with AgNO₃ even though ethylene productionwas inhibited. These results suggested that AgNO₃ may be havinganother more direct effect on protoplast release. One such sitemay be the cell wall or on cell metabolism conditioning cellsto release protoplasts after enzyme treatment. Key words: Sugarcane, cell suspension, protoplast, silver nitrate, ethylene     

Growth and quaternary alkaloid production in differentiating and non-differentiating strains of Ruta graveolens

Cultures of Ruta graveolens have been grown and maintained on Murashige and Skoog's medium supplemented with α-naphthalene acetic acid (1 mg/l.) and kinetin (0.1 mg/l.) or 2,4-dichlorophenoxyacetic acid (1 mg/l.), and glucose (30 g/l.). The production of dihydrofuroquinoline alkaloids has been investigated in these cultures. The present study showed that: (a) cultures of Ruta produced both differentiating and non-differentiating strains; (b) quaternary alkaloids (platydesminium, ribalinium and rutalinium) were present in all the strains and maximum contents were detected at 3-4 weeks growth; (c) high platydesminium and rutalinium content was associated with differentiation and callus formation, respectively; (d) changed concentrations of quaternary alkaloids were recorded in all the five strains grown on media supplemented with tryptophan and precursors (anthranilic acid, 5-methylanthranilic acid and 2,4-dihydroxyquinoline).     

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.     

Growth stimulation by catecholamines in plant tissue/organ cultures

Addition of catecholamines at micromolar concentrations caused a dramatic stimulation of growth of tobacco (Nicotiana tabacum) thin cell layers (TCLs) and Acmella oppositifolia “hairy” root cultures. A threefold increase in the rate of ethylene evolution was observed in the catecholamine-treated explants. Aminooxyacetic acid and silver thiosulfate, inhibitors of ethylene biosynthesis and action, respectively, reduced the growth-promoting effect of dopamine. However, these compounds alone could also inhibit the growth of the TCL explants. When ethylene in the culture vessel was depleted by trapping with mercuric perchlorate, dopamine-stimulated growth was still obtained, suggesting that ethylene does not mediate the dopamine effect. Dopamine potentiated the growth of TCLs grown in Murashige and Skoog medium supplemented with indoleacetic acid (IAA) and kinetin. When IAA was replaced by 2,4-dichlorophenoxyacetic acid, dopamine addition showed no growth-promoting effect. Instead, 2,4-dichlorophenoxyacetic acid stimulated the growth of TCL explants to the same extent as that obtained with IAA plus dopamine. Because synthetic auxins do not appear to be substrates for IAA oxidizing enzymes, we hypothesized that catecholamines exert their effect by preventing IAA oxidation. Consistent with this explanation, dopamine (25 micromolar) inhibited IAA oxidase activity by 60 to 100% in crude enzyme extracts from tobacco roots and etiolated corn coleoptiles, but had no effect on peroxidase activity in the same extracts. Furthermore, addition of dopamine to TCL cultures resulted in a fourfold reduction in the oxidative degradation of [1-¹⁴C]IAA fed to the explants. Because the growth enhancement by catecholamines is observed in both IAA-requiring and IAA-independent cultures, we suggest that these aromatic amines may have a role in the regulation of IAA levels in vivo.     

The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L.

Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

The influence of ethylene on proliferation and growth of rose shoot cultures

Ethylene accumulation in four different rose in vitro culture containers was evaluated. Multiplication rate was the highest, and axes most elongated, in the two containers where ethylene accumulation was limited. Pulse treatments of ethylene at various concentrations enhanced proliferation depending on concentration (5 ppm generally was the most favourable) and time of application, while reducing elongation of the shoots. An ethylene trap in the flask atmospheres of the cultures reduced rose shoot proliferation rate but increased elongation of the axes. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (CoCl₂), increased multiplication rate by providing a higher number of axes of a suitable size for subculture. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) had a beneficial effect on multiplication rate, although reducing longitudinal growth of the axes.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BA benzyladenine - GA₃ gibberellic acid - IBA indolyl-3-butyric acid     

Roots as an enhancing factor for the production of artemisinin in shoot cultures of Artemisia annua

Artemisinin was produced in differentiated shoot cultures of Artemisia annua L. but was undetected in callus or cell cultures. The growth regulators benzyladenine, kinetin, chlormequat, and daminozide, at concentrations which severely reduced rooting, reduced artemisinin production. A highly significant correlation (1% level) was observed between shoot artemisinin content and number of roots (r=0.775^**), but shoot number and artemisinin content were unrelated (r=-0.198). Benzyladenine increased shoot proliferation at 0.5 and 5.0 M, but decreased root production at 0.5, 5.0, and 50 M. The highest levels of artemisinin production (0.287% DW) were obtained in hormone-free medium when root production was maximized. Removal of roots from shoots cultured in hormone-free liquid medium reduced shoot artemisinin by 53% and shoot arteannuin B by 60%. Neither artemisinin, arteannuin B, or artemisinic acid were detected from roots developed in semi-solid or liquid medium.Abbreviations BA benzyladenine - CCC chlormequat - DW dry weight - FW fresh weight - GA₃ gibberellic acid - GC/MS gas chromatography/mass spectrometry - HPLC-EC high-performance liquid chromatography with electrochemical detection - MS Murashige & Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid Journal paper no. 14558 of Purdue Agricultural Research Progress     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Effect of ethylene on sanguinarine production from Papaver somniferum cell cultures

A Papaver somniferum cell line capable of producing sanguinarine equivalent to 3% of cell dry weight was used to determine if ethylene was involved in signalling the biosynthesis of this alkaloid. A 3.3-fold increase in ethylene emanation from these cell suspension cultures was observed 7 h after elicitation with a Botrytis fungal homogenate. The rate of ethylene release then decreased to near zero after 48 h, suggesting that a pulse of ethylene production may be involved in sanguinarine production. However, sanguinarine biosynthesis was not promoted when either the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), or the ethylene releasing agent, 2-chloroethylphosphonic acid (ethephon), was added to the culture. These results strongly suggest that ethylene is not intimately involved in the production of sanguinarine from Papaver somniferum cell cultures or in the transduction of the elicitation event.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid     

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l^–1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l^–1.     

Effects of light,phosphate, and oxygen on ethylene formation and conidiation in surface cultures ofpenicillium cyclopium westling

Penicillium cyclopium growing in a surface culture with 2-ketoglutarate or glutamate as a sole carbon source produced ethylene in two phases. The first peak of ethylene production (EP 1) was associated with aerial mycelium growth whereas the second peak of ethylene production (EP 2) occurred with formation and maturation of conidia. Conidiation was induced by blue light between 120 and 172 h after the culture was started and depended on the presence of a carbon source at the stage of conidiophore initiation. Exogenous phosphate content dropper rapidly before the onset of conidiation. The EP 2 was connected with conidiation via this drop. Addition of phosphate prior to the conidiophore initiation and during conidiation inhibited EP 2 without affecting conidiation, but conidia lacked a green pigment and their germination ability decreased by 905. Exogenous ethylene did not restore normal development. The EP 2 in asporogenic cultures was evoked by incubation in the dark and by phosphate removal. The EP 2 and conidiation were accompanied by an increased oxygen consumption. The EP 1 yield of ethylene depended only on biomass growth and was unaffected by any treatment mentioned above.     

Hormone independent root organ cultures of rye (Secale cereale)

This work describes the growth of rye root organ cultures which were capable of being repeatedly subcultured in hormone-free medium. They showed morphological characteristics, growth rate, inability to produce shoots, and response to auxins and cytokinins similar to those of the Agrobacterium rhizogenes (Ri plasmid) transformed hairy root cultures of tobacco and red beet which were used for comparison. The root cultures of rye were initiated from callus produced on a medium containing the growth regulators (plant hormones) 2,4-d and kinetin, then transferred to hormone-free medium. However not all rye explants gave rise to callus that would differentiate into stable hairy root cultures and rye seedling root explants did not grow if placed directly on a hormone-free medium. Rice and wheat produced callus and roots on a medium containing hormones but root organ cultures could not be maintained on a hormone-free medium.     

Effect of aeration on ethylene production by soil bacteria and soil samples cultivated in a closed system

Summary Ethylene production was studied in shaken cultures ofPseudomonas putida andPseudomonas fluorescens isolated from soil and in unsterile garden soil samples moistened to 60% of the water holding capacity. The highest ethylene accumulation in bacterial cultures was reached under conditions of delayed aeration,i.e. when the culture was closed and the aeration started after the oxygen content decreased to 4%. The ethylene production rose immediately after the beginning of aeration. Under these conditions ethylene production was inP. fluorescens 2–3 times and in glucosecultivatedP. putida 6 times higher than in the fully aerated cultures. Methionine stimulated ethylene production byP. fluorescens, whereas glucose proved to be more suitable forP. putida. This strain was incapable of growth on methionine as the sole carbon source. Samples of nonsterile garden soil produced the highest amounts of ethylene under anaerobic conditions. Artificial inoculation of soil samples byP. putida resulted in an increase of ethylene formation in samples with delayed aeration. Addition of glucose or glucose with methionine stimulated ethylene production in all soil samples.     

Effects of auxins and cytokinins on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis

Cell suspension cultures of Anchusa officinalis required exogenous phytohormones for their normal growth. Cell lysis was observed at the third passage in a hormone-free medium. Using hormone — depleted cells, the effects of auxins (2,4-D, NAA, IAA and CFP) and cytokinins (BA, kinetin, and zeatin) on cell growth and RA production were investigated. All auxins tested could maintain growth and integrity of the cells whereas cytokinins alone could not, suggesting that this culture is auxindependent. Among the auxins tested, NAA had a pronounced effect on RA production. The total RA content obtained at optimum NAA concentration (0.25 mg/l) reached 1.7 g/l (12% of dry weight). The kinetics of growth and RA production suggested that the increase in final RA content was due to both an increase in the rate of RA synthesis and initiation of the period of synthesis in the exponential rather than the linear growth phase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - CFP 2-chloro-4-fluorophenoxyacetic acid - BA 6-benzyladenine - RA rosmarinic acid     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Growth dynamics and domoic acid production of Pseudo-nitzschia sp. in response to oil and dispersant exposure

The diatom genus Pseudo-nitzschia is a common component of phytoplankton communities in the Gulf of Mexico and is potentially toxic as some species produce the potent neurotoxin domoic acid. The impact of oil and chemical dispersants on Pseudo-nitzschia spp. and domoic acid production have not yet been studied; preliminary findings from a mesocosm experiment suggest this genus may be particularly resilient. A toxicological study was conducted using a colony of Pseudo-nitzschia sp. isolated from a station off the coast of Louisiana in the Gulf of Mexico. The cultures were exposed to a water accommodated fraction (WAF) of oil and a diluted chemically enhanced WAF (DCEWAF) which was a mix of oil and dispersant (20:1). Exposure to WAF induced a lag phase but did not inhibit growth rates once in exponential growth. Cultures grown in DCEWAF did not experience a lag phase but had significantly lower growth rates than the Control and WAF cultures. The cellular quota of domoic acid was higher in cultures treated with DCEWAF and WAF relative to their control values, and half of the domoic acid had leaked out of the cells into the surrounding seawater in the DCEWAF cultures while all the domoic acid remained inside the cells in WAF-treated cultures. These results suggest that the presence of oil could lead to toxic blooms, but that the application of dispersant could decrease bioaccumulation of domoic acid through the food web.     

Quinine and quinidine production by cinchona leaf,root and unorganized cultures

Serially propagated Cinchona ledgeriana and C. succirubra (Rubiaceae) leaf, root and unorganized suspension cultures established from germinated seeds were studied for quinine and quinidine production. Leaf organ cultures were grown and subcultured in Murashige and Skoog's Revised Tobacco Medium supplemented with benzyladenine; root organ cultures were grown on the same medium supplemented with indolebutyric acid; and unorganized suspension cultures were grown on the same medium supplemented with 2,4-dichlorophenoxyacetic acid and benzyladenine. On a dry weight basis, leaf organ cultures of C. ledgeriana contained 0.06 % quinine and 0.05 % quinidine and of C. succirubra contained 0.04 % quinine and 0.04 % quinidine. No quinine and quinidine were detected in either root organ or unorganized suspension cultures.     

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1?mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1?mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5?mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1?mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8???g?ml^?1 for the HL-60, HeLa, and MCF-7 cells, respectively.