The cytogenetic effect of natural mutagenesis modifiers in a human lymphocyte culture. The action of aminobenzamide during the gibberellic acid induction of chromosome aberrations

The 3-aminobenzamide sensibilization of the cytogenetic activity of gibberellic acid in the culture of human lymphocytes has been investigated. Two-three-fold increase of the chromosome aberrations induced by gibberellic acid, irrespective of the time of 3-aminobenzamide addition to the cultures (the 28th, 46th, 72th hrs of the cultivation) is shown.     

Obligate methylotroph <Emphasis Type="Italic">Methylobacillus arboreus</Emphasis> Iva<Superscript>T</Superscript> synthesizes a plant hormone,gibberellic acid GA<Subscript>3</Subscript>

An obligate methylotroph Methylobacillus arboreus Iva^Т (VKM B-2590^Т, CCUG 59684^T, DSM 23628T) is the first known aerobic methylotrophic bacterium capable of synthesis of the bioactive gibberellic acid GA₃. Primary separation and identification of gibberellic acid from the culture liquid of methanol-grown culture were carried out using thin-layer chromatography and high-performance liquid chromatography. The concentration and structure of the gibberellic acid GA₃ were determined by liquid chromatography?mass spectrometry (LC/MS). Biological activity of the isolated compound was confirmed by tests on sprouts of lettuce (Laсtuca sativa L.).     

Effect of Auxin and Gibberellin on Sporulation in Yeast

Effect of auxin and gibberellic acid on sporulation of a yeast, Saccharomyces ellipsoideus, was studied. When added to the sporulation media, gibberellic acid promoted sporulation. The sporulation rate was higher in the medium SGV with vitamins than in the vitamin-free SG, but the effect of gibberellic acid was more pronounced in the latter. Auxin (IAA, 2,4-D, and NAA) inhibited sporulation in SGV, but promoted it in SG. This sporulation-promoting effect of IAA was reversed by an antiauxin, 2,4,6-T. Preculturing in the presence of added IAA increased sporulation. Added to the preculture medium, gibberellic acid alone showed little effect on sporulation, but in combination with IAA it enhanced sporulation conspicuously. IAA and gibberellic acid were effective in sporulation promotion only when added before the nuclear enlargement occurred in sporulation culture.     

Regulation of invertase levels in Avena stem segments by gibberellic Acid, sucrose, glucose, and fructose

Gibberellic acid and sucrose play significant roles in the increases in invertase and growth in Avena stem segments. About 80% of invertase is readily solubilized, whereas the rest is in the cell wall fraction. The levels of both types of invertase change in a similar manner in the response to gibberellic acid and sucrose treatment. The work described here was carried out with only the soluble enzyme. In response to a treatment, the level of invertase activity typically follows a pattern of increase followed by decrease; the increase in activity is approximately correlated with the active growth phase, whereas the decrease in activity is initiated when growth of the segments slows. A continuous supply of gibberellic acid retards the decline of enzyme activity. When gibberellic acid was pulsed to the segments treated with or without sucrose, the level of invertase activity increased at least twice as high in the presence of sucrose as in its absence, but the lag period is longer with sucrose present. Cycloheximide treatments effectively abolish the gibberellic acid-promoted growth, and the level of enzyme activity drops rapidly. Decay of invertase activity in response to cycloheximide treatment occurs regardless of gibberellic acid or sucrose treatment or both, and it is generally faster when the inhibitor is administered at the peak of enzyme induction than when given at its rising phase. Pulses with sucrose, glucose, fructose, or glucose + fructose elevate the level of invertase significantly with a lag of about 5 to 10 hours. The increase in invertase activity elicited by a sucrose pulse is about one-third that caused by a gibberellic acid pulse given at a comparable time during mid-phase of enzyme induction, and the lag before the enzyme activity increases is nearly twice as long for sucrose as for gibberellic acid. Moreover, the gibberellic acid pulse results in about three times more growth than the sucrose pulse. Our studies support the view that gibberellic acid, as well as substrate (sucrose) and end products (glucose and fructose), play a significant role in regulating invertase levels in Avena stem tissue, and that such regulation provides a mechanism for increasing the level of soluble saccharides needed for gibberellic acid-promoted growth.     

Production of cell wall hydrolyzing enzymes by barley aleurone layers in response to gibberellic Acid

The cell walls of barley (Hordeum vulgare var. Himalaya) aleurone layers undergo extensive degradation during the tissue's response to gibberellic acid. Previous work had shown that these cell walls consist almost entirely of arabinoxylan. In this study we show that gibberellic acid stimulates endo-β-1,4-xylanase activity in isolated aleurone layers. In addition, gibberellic acid enhances the activity of two glycosidases: β-xylopyranosidase and α-arabinofuranosidase. No gibberellic acid-stimulated cellulase activity was detected. Germination studies showed a similar pattern of enzyme development in intact seeds.     

Reduction of alpha-amylase by an inhibitor from carob

Inhibitor C, one of the inhibitory fractions of the extract of Carob fruit, reduces the amount of α-amylase in the culture medium of endosperm halves of barley seed which have been treated with gibberellic acid as compared with seed halves treated with gibberellic acid alone. A similar reduction is found in the α-amylase produced by embryo halves of barley seed which have not been treated with gibberellic acid.     

Actions of gibberellic Acid and phytochrome on the germination of grand rapids lettuce seeds

Red light and gibberellic acid were about equally effective in promoting germination of Grand Rapids lettuce (Lactuca sativa L.) seeds. With initial far red light treatment more than 80% remained dormant in subsequent dark storage. After 2 days of dark storage, red light effectively promoted germination, while gibberellic acid action was weak. With between 2 and 10 days of dark storage, gibberellic acid had little effect, while promotion by red light decreased slowly and finally disappeared. After 10 days of dark storage, both gibberellic acid and red light were required for germination. The dark storage treatment interferes with phytochrome-independent germination processes and cannot be overcome by added gibberellic acid. However, storage may also decrease the effectiveness of endogenous gibberellins. Phytochrome-dependent germination seems to require only low levels of endogenous gibberellin activity or the addition of gibberellic acid. Gibberellins and red light appear to act on germination by regulation of sequential sites of a branched-looped pathway.     

Influence of different concentrations of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale

In this work, the effect of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale was studied. The study of androgenesis was conducted on two model samples YaTKh-327-11 and YaTKh-18-11. The research revealed that the addition of the antioxidant ascorbic acid at a concentration of 2–8 mg/L into the culture medium to induce embryogenesis enhanced the formation of androgen structures and regeneration of green plants. The addition of gibberellic acid into the culture medium to induce embryogenesis gave negative effects. The average yield of green plants in the anther culture of spring triticale was 2 pcs/100 anthers. According to the research, 277 green plants were received and spontaneous doubling of chromosomes was recorded in 26.7%.     

The influence of gibberellic acid and abscisic acid on cell and tissue differentiation of bean callus.

Bean callus was induced to form roots (tissue differentiation) and vascular nodules (cell differentiation) by lowering the ratio of auxin to cytokinin in the growth medium. Both types of differentiation were inhibited by the addition of abscisic acid (at concentrations greater than I muM) to induction medium. Initiation of differentiation was inhibited, but its subsequent development was not, and the inhibition was not affected by the addition of gibberellic acid. Addition of gibberellic acid (GA) alone to induction medium stimulated tissue differentiation, although cell differentiation was unaffected (30 muM GA) or inhibited (45 muM GA) and its onset was delayed at both concentrations. Root initiation was also stimulated by gibberellic acid (0.I-45 muM) at an auxin-to-kinin ratio 10 times that normally optimal for cell differentiation. The phenylalanine ammonia lyase (PAL) activity of the calluses was closely correlated with the amount of cell differentiation which had occurred, and measurement of this confirmed that gibberellic acid delayed the initiation of cell differentiation. The increase and subsequent decline of PAL and betaI leads to 3 glucan synthetase activities, normally induced by transfer to induction medium, was abolished by abscisic acid. Addition of gibberellic acid did not affect the betaI leads to 3 glucan synthetase activity.     

Callus formation versus differentiation of cultured barley embryos: hormonal and osmotic interactions

The effect of various plant growth substances as single agents was evaluated in a complex tissue culture system: whole embryo culture of early differentiating barley embryos. Callus formation in unsupplemented medium derives from the mesocotyl and is uniquely characteristic of cultures initiated at this stage of embryonic development. This phenomenon could be prevented or reversed by incorporation of gibberellic acid in the medium resulting in plantlet formation. Indoleacetic acid enhanced callus growth, whereas kinetin did not promote either callus or meristematic development. Callus tissue markedly accumulated starch, effectively lowering the cellular osmolarity, while inducing a corresponding rise in the osmolarity of the culture medium. This osmotic pattern was reversed by gibberellic acid induction of shoot formation. These osmotic-hormonal interactions are interpreted relative to in vivo, in situ normal embryogeny or developmental lesions such as tumors.     

Ethylene effects on amylase activity from isolated barley aleurone layers : possible modification by proteolytic enzymes

The effect of protease inhibitors on the response of gibberellic acid-treated barley aleurone layers to ethylene was examined. In the absence of protease inhibitors, ethylene plus gibberellic acid initially increased the production of amylase activity relative to layers incubated with gibberellic acid alone. Exposure to ethylene plus gibberellic acid for 48 hours or longer, however, led to depressed levels of amylase activity compared to samples incubated with gibberellic acid in hydrocarbon-free air.

The direct assay of proteolytic activity revealed a small increase in activity in response to ethylene. The significance of this response was probed further by including inhibitors of barley proteases in the incubation medium. When potassium bromate was introduced, ethylene did not cause any alteration in amylase activity compared to samples incubated in hydrocarbon-free air. However, in the presence of N-ethylmaleimide, ethylene treatment induced a 52% increase in amylase activity recovered from samples after 48 hours. These results suggest that proteases contribute to the loss of amylase activity in response to ethylene and thus alter the apparent effect of ethylene on amylase synthesis. The effect of protease inhibitors on other hydrolases is also discussed.

During the incubation period, the pH of the medium declined significantly. However, ethylene had no effect on the extent of this decline.

     

Effect of red light and gibberellic acid on lipid metabolism in germinating spores of the fern Anemia phyllitidis

The spores of the fern Anemia phyllitidis contain abundant quantities of lipid as reserve material. Germination of these spores can be induced either by red light or, even in the dark, by gibberellic acid. The effects of both these factors on lipid degradation, lipase and isocitrate lyase activities, and on the fatty acid composition have been studied in the course of the germination process. During germination in darkness with gibberellic acid, the fatty acid composition remained similar to that in the ungerminated spore. In contrast, when spores were germinated in red light, α-linolenic acid was synthesized. Little activity of lipase and isocitrate lyase could be detected in the dry spore. Red light or gibberellic acid affected a dramatic increase of the activities of these enzymes. Lipid breakdown and lipase activity were more active in red light, however. Permanent stimuli were necessary for growth and complete lipid degradation. Induction of germination simultaneously with both factors revealed an additivity of the effects of red light and gibberellic acid.     

Synergism between gibberellic acid and low Pfr levels inducing germination of Kalanchoë seeds

Sown on water, seeds of Kalanchoëbiossfetdiana Poelln. cv. Feuerblute are absolutely light-requiring and show full red/far-red reversibility. In seeds, sown on 2 ×10^-3 M gibberellic acid, red/far-red reversibility disappears and both short red and far-red irradiations induce germination. Gibberellic acid alone does not induce germination, but it increases the physiological activity of P_fr to the extent, that the low P_fr level obtained by far-red irradiation becomes very effective. The synergism between gibberellic acid and far-red light appears after a two-day incubation; period. The nature of this lag phase was examined by measuring both germination and uptake of labelled gibberellic acid in intact seeds and seeds with a punctured seed coat. The lag phase was shown to be independent of the uptake kinetics of gibberellic acid and allows development to a specific stage, necessary for germination after phytochrome-phototransformation. The kinetics of the uptake of gibberellic acid by intact seeds and embryos of intact seeds are different. In intact seeds most of the gibberellic acid is retained in the seed coat; only a small fraction actually penetrates to the embryo where it can exert its physiological activity.     

Stimulation of the gibberellic acid synthesis by Aspergillus niger in submerged culture using a precursor

Stimulation of Aspergillus niger in submerged culture using a commonly known precursor, mevalonic acid (MVA), was investigated in terms of growth and gibberellic acid production. Increasing concentrations of MVA up to 60 M enhanced product and growth yields. Above this amount, gibberellic acid yields and growth were gradually decreased.     

Callus formation versus differentiation of cultured barley embryos: Hormonal and ostomic interactions

Summary The effect of various plant growth substances as single agents was evaluated in a complex tissue culture system: whole embryo culture of early differentiating barley embryos. Callus formation in unsupplemented medium derives from the mesocotyl and is uniquely characteristic of cultures initiated at this stage of embryonic development. This phenomenon could be prevented or reversed by incorporation of gibberellic acid in the medium resulting in plantlet formation. Indoleacetic acid enhanced callus growth, whereas kinetin did not promote either callus or meristematic development. Callus tissue markedly accumulated starch, effectively lowering the cellular osmolarity, while inducing a corresponding rise in the osmolarity of the culture medium. This osmotic pattern was reversed by gibberellic acid induction of shoot formation. Thise osmotic-hormonal interactions are interpreted relative to in vivo, in situ normal embryogeny or developmental lesions such as tumours. Research supported by Inst. Grant No. 994.     

Survival of shoot meristems of tomato seedlings frozen in liquid nitrogen

An explant containing the primary shoot meristem was dissected from intact tomato seedlings after thawing from liquid nitrogen. Surviving explants produced shoots directly by normal meristem growth when cultured in the presence of gibberellic acid. Without gibberellic acid all surviving explants produced callus tissue and subsequently adventitious shoots, with no direct outgrowth of the primary meristem.Dimethyl sulphoxide (15%) in culture medium and a cooling rate changing continuously from 20 to 55 °C min^?1 between 0 and ?120 °C were required for optimal survival.Nonfrozen material produced shoots directly without the requirement for gibberellic acid indicating that hormonal regulation of organised growth by the shoot meristem had been altered by the freeze/ thaw process.     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.     

Effect of exogenous gibberellic acid,abscisic acid,and benzylaminopurine on epicotyl dormancy of cultured herbaceous peony embryos

Epicotyl dormancy was broken in cultured peony (Paeonia lactiflora Pall.) embryos after topical application of agarose gels containing gibberellic acid, with optimum growth at 1.5 mM gibberellic acid. Addition of 100 M abscisic acid to the medium resulted in complete inhibition of gibberellic acid-stimulated promotion of dormant epicotyls. Epicotyl dormancy was also broken in embryos by culture on media containing 1 or 10 M benzylaminopurine. A highly significant increase in leaf number occurred when embryos were both cultured on medium containing benzylaminopurine and treated topically with gibberellic acid. Anatomical and morphological studies indicated that the increase in shoot growth was due to the development and growth of 1) buds formed at the cotyledonary node, 2) axillary buds, and 3) adventitious meristems originating from subepidermal parenchymatous tissue.Abbreviations ABA abscisic acid - BA N⁶-benzylaminopurine - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - LS Linsmaier and Skoog     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Starch Metabolism, Respiration, and Shoot Formation in Tobacco Callus Cultures

The starch content of shoot-forming and non-shoot-forming tobacco callus cultured in light and darkness was determined. A variety of carbohydrates and cytokinins incorporated into the culture medium were effective in bringing about starch accumulation and shoot formation in the tissue. In addition, the respiratory activity of the callus, grown in the presence or absence of gibberellic acid, was measured. A strong correlation between the starch content of the tissue, its rate of respiration, and shoot formation was observed.