Influence of growth retardants on the internode and ethylene production of sunflower plants

The growth-retarding activity of the norbornenodiazetine tetcyclacis and the di-oxanylalkenyl triazole LAB 150 978 as well as the ethylene-forming compounds 2-chloroethyl-phosphonic acid (ethephon) and 1-amino-cyclopropane-l-carboxylic acid (ACC) on stem histogenesis and ethylene production of sunflower plants ( He-lianthus annuus L. cv.Spanners Allzweck) has been studied. The shoot growth of plants hydroponically grown and treated was reduced by the compounds. The shortening in the length of the 1st internode caused by tetcyclacis and LAB 150 978 was mainly induced by inhibition of cell division (the internode possessed fewer cortical cells per cell file). In contrast, ethephon and ACC decreased internode elongation mainly by reducing the rate of cell enlargement.
The ethylene production of sunflower seedlings cultivated on agar nutrient medium rose with increasing concentrations of ethephon and ACC, the shoot length of the plants being progressively reduced.
Tetcyclacis and LAB 150 978 inhibited both the formation of ethylene and shoot growth. It is suggested that in contrast to ethephon and ACC, tetcyclacis and LAB 150 978 do not achieve their growth-retarding effect by influencing the production of ethylene.     

Inhibition of ethylene production in sunflower cell suspensions by the plant growth retardant BAS 111..W: Possible relations to changes in polyamine and cytokinin contents

At a concentration of 10^–5 mol · L^–1 the triazole-type growth retardant BAS 111..W completely inhibited the transiently elevated ethylene production in the exponential growth phase of heterotrophic sunflower cell suspensions. This effect, which could not be restored by adding gibberellin A₃, was accompanied by transiently increased levels of 1-aminocyclopropanecarboxylic acid (ACC) in the cells, which was increasingly converted to N-malonyl-ACC. Thus, the reactions from ACC to ethylene catalyzed by the ethylene-forming enzyme appeared to be blocked by the retardant. Concomitantly, higher endogenous levels of free spermidine and particularly spermine were found over control, whereas free putrescine, the direct precursor of both polyamines, simultaneously decreased. We assume that the remaining S-adenosylmethionine from ethylene biosynthesis was increasingly incorporated into spermidine and finally spermine. A further relation appears to exist between the reduced ethylene production and enhanced levels of cytokinins in the cells. The application of both BAS 111..W and aminoethoxyvinylglycine depressed ethylene formation while immunoreactive cytokinins from isopentenyladenosine-, trans-zeatin ribo-side-, and dihydrozeatin riboside-type increased. By additional treatment with ACC, the effects could partially be reversed. On the other hand, stimulation of ethylene production by ACC alone or ethephon considerably lowered cytokinin levels.     

Inhibition of ethylene production in sunflower cell suspensions by a novel oxime ether derivative

During the incubation of undifferentiated cell suspensions of sunflower (Helianthus annuus L. cv. Spanners Allzweck) ethylene production was effectively inhibited by the novel oxime ether derivative LAB 181 508, [[(Isopropyliden)-amino]-oxy]-acetic acid-2-(methoxyl)-2-oxoethylester (PACME). The compound was most active during the first 6 days of incubation exhibiting a value of 50% inhibition at 9.5×10^–6 mol×L^–1. The pattern of changes in the internal 1-aminocyclopropanecarboxylic acid (ACC) and N-malonyl-ACC (MACC) levels paralleled the influence on ethylene formation. While the addition of ACC fully restored ethylene production, applied S-adenosyl-L-methionine (SAM) was not effective. Experiments with [¹⁴C]indole-3-acetic acid (IAA) revealed that LAB 181 508 did not affect auxin uptake into suspension cells of sunflower. The results suggest that LAB 181 508 reduces ethylene formation by inhibiting the conversion of SAM to ACC in the biosynthetic pathway. In comparison to the structurally related inhibitor of ACC synthase, aminoethoxyvinylglycine (AVG), LAB 181 508 reduced growth and viability of the suspension cells only slightly. Low phytotoxicity of LAB 181 508 combined with a less complicated chemical synthesis might offer interesting aspects for physiological research and horticultural and agricultural practice.     

Inhibition of ethylene production in sunflower cell suspensions by a novel oxime ether derivative

During the incubation of undifferentiated cell suspensions of sunflower (Helianthus annuus L. cv. Spanners Allzweck) ethylene production was effectively inhibited by the novel oxime ether derivative LAB 181 508, [[(Isopropyliden)-amino]-oxy]-acetic acid-2-(methoxyl)-2-oxoethylester (PACME). The compound was most active during the first 6 days of incubation exhibiting a value of 50% inhibition at 9.5×10^?6 mol×L^?1. The pattern of changes in the internal 1-aminocyclopropanecarboxylic acid (ACC) and N-malonyl-ACC (MACC) levels paralleled the influence on ethylene formation. While the addition of ACC fully restored ethylene production, applied S-adenosyl-L-methionine (SAM) was not effective. Experiments with [¹⁴C]indole-3-acetic acid (IAA) revealed that LAB 181 508 did not affect auxin uptake into suspension cells of sunflower. The results suggest that LAB 181 508 reduces ethylene formation by inhibiting the conversion of SAM to ACC in the biosynthetic pathway. In comparison to the structurally related inhibitor of ACC synthase, aminoethoxyvinylglycine (AVG), LAB 181 508 reduced growth and viability of the suspension cells only slightly. Low phytotoxicity of LAB 181 508 combined with a less complicated chemical synthesis might offer interesting aspects for physiological research and horticultural and agricultural practice.     

Ethylene production by growing and senescing pear fruit cell suspensions in response to gibberellin

A pear (Pyrus communis L. cv Passe Crassane) cell suspension was used as a model system to study the influence of gibberellin on processes related to fruit ripening. Growth of the cell cultures was inhibited and their loss of viability was accelerated when 0.5 millimolar gibberllic acid (GA₃) was added to suspensions at two stages of cell development, namely, growth and quiescence. Cell respiration rate was unaffected up to 2 millimolar GA₃ but ethylene production, both basal and 1-aminocyclopropane-1-carboxylic acid-induced, was inhibited at all stages of cell development. However, the degree of inhibition decreased as the cell cultures aged. The site of ethylene inhibition by GA₃ appeared to be related to the ethylene-forming enzyme. The coincident acceleration of cell senescence and inhibition of ethylene production indicate that the pear cell suspension cannot serve as an analogous model for studying the mode of action of gibberellin in delaying ripening and senescence of fruits in its entirety, although certain specific effects might be relevant.     

A new micro-method for testing plant growth retardants in cell suspension cultures

A microtest sytem for detecting the growth retarding potential of chemosynthetic compounds in cell suspension cultures is presented. The new screening technique involves the cultivation of one millilitre of suspension in small sterile test tubes and indirect monitoring of growth by measuring conductivity changes in the medium. Highly significant correlations were obtained in experiments comparing the effects of a broad range of plant growth retardants on the growth of different suspensions in erlenmeyer flasks measured by cell counting and in test tubes with growth recorded by conductivity changes in the medium. The method described is suitable for all suspension cultures hitherto studied.For Abbreviations, see Materials and Methods     

Estimation of growth yield and maintenance coefficient of plant cell suspensions

Methodology is presented for the determination of growth yield (Y(g)) and maintenance coefficient (m) for carbon utilization of plant cells grown in suspension culture. Estimation of Y(g) and m requires measurements of specific growth rate (micro) and specific rate of substrate uptake (q) at different growth limiting substrate concentrations. Batch culture of tobacco cells did not permit evaluation of Y(g) and m because micro is constant and maximal during most of the growth cycle. In batch culture, the period of declining specific growth rate is extremely brief because of the rapid transition from logarithmic growth to stationary phase. This occurs because the K(m) for growth is relatively small compared to the initial sucrose concentration. Thus, when the substrate level reaches the K(m), the large mass of cells rapidly depletes the remaining substrate. In contrast, semicontinuous culture facilitates the determination of Y(g) and m because various steady-state growth rates can be achieved. Mathematical expressions were developed to determine the effective values of micro and q over the semicontinuous replacement interval. The validity of this approach was verified by conducting simulations using experimentally determined parameters.     

Effects of growth retardants on bulbil production by Narcissus twin-scales

Narcissus twin-scale propagules, cut from various positions in the parent bulb, were incubated with gibberellic acid (GA₃) or growth retardants. Bulbil production was inhibited in all cases by GA₃ at 10–100 mg/litre. Chlormequat chloride, chlorphonium chloride and paclobutrazol also inhibited bulbil production; in the case of paclobutrazol, outer twin-scales were more sensitive to lower concentrations of the retardant than inner ones. Ancymidol enhanced bulbil production (in numbers by up to 15%) in the outermost twin-scales, and had no stimulatory effect on the inner twin-scales. Treatments affected bulbil numbers and bulbil lengths about equally.     

Comparative potency of different plant growth retardants in cell cultures and intact plants

A comparison of the efficiency of a broad range of plant growth retardants on cell division growth of 13 cell suspension cultures is presented. The results show that (1) the new plant bioregulator tetcyclacis (NDA) is the compound with the highest activity in inhibiting cell division of all cultures tested, and (2) cell cultures react species-specifically to various compounds. Significant correlations between the results from suspension cultures and intact seedlings of the same plant species demonstrate the usefulness of cell cultures for identifying substances with a growth-regulating potency. Futhermore, the usefulness of cell cultures for establishing structure-activity relationships was shown with structural analogues of chlormequat and mepiquat chloride.     

Comparative potency of different plant growth retardants in cell cultures and intact plants

A comparison of the efficiency of a broad range of plant growth retardants on cell division growth of 13 cell suspension cultures is presented. The results show that (1) the new plant bioregulator tetcyclacis (NDA) is the compound with the highest activity in inhibiting cell division of all cultures tested, and (2) cell cultures react species-specifically to various compounds. Significant correlations between the results from suspension cultures and intact seedlings of the same plant species demonstrate the usefulness of cell cultures for identifying substances with a growth-regulating potency. Futhermore, the usefulness of cell cultures for establishing structure-activity relationships was shown with structural analogues of chlormequat and mepiquat chloride.     

The effect of growth retardants on anthocyanin production in carrot cell suspension cultures

The effect of growth retardants on anthocyanin production was studied in wild carrot (Daucus carota) cell suspension cultures. Paclobutrazol [(2RS,3RS) — 1 — (4-chlorophenyl) — 4,4 —dimethyl-2-(1,2,4-triazol-1-yl) pentan-3-ol], uniconazole [(E)-1-(4-chlorophenyl-4,4 —) dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol], tetcyclacis [5-(4-chloro-phenyl) -3,4,5,9,10-pentaaza-tetracyclo-5, 4, 10^2,6, O^8,11 — dodeca-3, 9-diene], ancymidol [-cyclopropyl — 4 — methoxy-(pyrimidine-5-yl)benzyl alcohol] and CCC (2-chloro-ethyltrimethylammonium chloride) increased anthocyanin accumulation. AMO-1618 [(2-isopropyl-5-methyl-4-trimethyl-ammonium-chloride)-phenyl-1-piperidinium carboxylate] did not increase anthocyanin accumulation in the first passage but did increase it during the second passage on medium for improved anthocyanin accumulation. Prohexadione (3,5-dioxo-4-propionylcyclohexane carboxylic acid) decreased anthocyanin accumulation by 10%–12.5%.The inhibitory effect of gibberellin on anthocyanin accumulation was reversed by paclobutrazol. Paclobutrazol together with 10^–6M GA₃ increased anthocyanin level from 33% of control in GA₃ treated cell suspension to 76%. These results are consistent growth retardants increasing anthocyanin accumulation in carrot cell suspension cultures by inhibiting gibberellin biosynthesis.     

The action of plant growth retardants on terpenoid biosynthesis

Summary The plant growth retardants (2-chloroethyl)-trimethylammonium chloride (CCC) and 2-isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl-piperidine-1-carboxylate (AMO-1618) inhibit gibberellic-acid biosynthesis inFusarium moniliforme at the cyclisation of geranylgeraniol to (-)-kaurene, causing an accumulation of geranylgeraniol. The two inhibitors have no effect on the biosynthesis of ergosterol inF. moniliforme or sitosterol in barley seedlings.     

Effect of bacitracin on growth and monoclonal antibody production by tobacco hairy roots and cell suspensions

Hairy roots and suspended cells of transformedNicotiana tabacum were used to produce full length murine IgG₁ monoclonal antibody. The maximum amount of antibody accumulated per g dry weight in the hairy root cultures was 6.5 times that in the suspension cultures. Up to 48% of the antibody in the suspension cultures was found extracellularly, while a maximum of only 17% was recovered from the hairy root medium. The amount of assembled antibody in the root and suspension cultures was significantly reduced by intracellular and/or extracellular antibody degradation soon after the end of the exponential growth phase. Bacitracin, a polypeptide antibiotic, has been shown in previous work to prevent degradation of peptides and hormones in plant and mammalian systems. Treatment of hairy roots and cell suspensions with 100 μg/mL bacitracin was not sufficient to prevent loss of antibody from the cultures, but improved the specific growth rates by up to 53%. At concentrations of 250 μg/mL and above, bacitracin had a toxic effect on hairy roots, which may limit the application of this peptide in plant tissue culture.     

Regulation of isopentenoid biosynthesis by plant growth retardants in Nicotiana tabacum

The effects of AMO-1618, AY-9944 and SKF-7997 on the growth of Nicotiana tabacum and on the biosynthesis of acidic and neutral isopentenoids in the seedlings were examined. All three compounds significantly retarded stem elongation but not dry wt of the plants. Incorporation of radioactivity from [2- ¹⁴C]-mevalonate into acidic and neutral isopentenoids showed a direct relationship between the radioactivity in the two fractions and stem growth. All three compounds inhibited steps before as well as after squalene formation, but the acylic and polycyclic isopentenoids which accumulated as a result of the inhibition were not necessarily the same in each of the treatment groups. This indicates that stem growth is probably influenced not by a single product of isopentenoid biosynthesis, but rather by several products, which may even act independently in their effects on developmental processes.     

Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.