Diheteroarylurea derivatives as adventitious rooting adjuvants in mung bean shoots and M26 apple rootstock

The present research investigates the biological profile of eight symmetrical diheteroarylureas and phenylheteroarylureas, testing their hypothetical cytokinin-like activity and rooting activity. Cytokinin-like activity was assayed by the betacyanin (so-called amaranthin) accumulation test and by the tomato regeneration test. The rooting activity was assessed using the mung bean rooting test, the apple stem slice test and the rooting of apple microcuttings. Three compounds, 1,3-di(pyrazin-2-yl)urea (3a), 1,3-di(benzo[d]oxazol-5-yl)urea (3b) and 1,3-di(benzo[d]oxazol-6-yl)urea (3c), enhanced adventitious root formation in apple stem slice test, but only 3b and 3c were active in the mung bean rooting test. Compound 3b, that showed the best rooting activity, was also able to enhance the adventitious root formation in apple microcuttings. None of the compounds showed cytokinin-like activity.     

Effect of Cl-Substitution on Rooting- or Cytokinin-like Activity of Diphenylurea Derivatives

Twenty-eight Cl-substituted diphenylurea derivatives differing in either the number and the position of the substituents, or in the type of substitution, that is, symmetric or asymmetric, were synthesized. Their hypothetical enhancement of rooting activity was assayed using the mung bean shoot bioassay; their possible cytokinin-like activity was assessed using the betacyanin (so-called amaranthin) accumulation test and the tomato regeneration test. Seven Cl-substituted diphenylurea derivatives (2E, 4A, 4B, 4E, 4G, 6A, 6B) having two substituted phenyl rings showed the capacity to enhance adventitious root formation in mung bean shoots. Furthermore the presence of a halogen substituent was not sufficient to reach the adventitious rooting activities shown by the N,N -bis-(2,3-methylenedioxyphenylurea) and the N,N -bis-(3,4-methylenedioxyphenylurea), two diphenylurea derivatives for which an interaction with auxin was the first reported in enhancing adventitious root formation. Seven compounds (1B, 3E, 3D, 4B, 4E, 4F, 6B) showed cytokinin-like activity and three of them (4B, 4E, 6B) also evidenced rooting activity, once more demonstrating the wide action spectrum of diphenylurea derivatives.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: II. Auxin- and Abscisic Acid-like Activity

The wilty tomato mutant, flacca, and the control variety, Rheinlands Ruhm, were compared with regard to the endogenous activity and concentration of auxin- and abscisic acid-like substances during ontogeny. The mutant wilts fast under water deficit because of inability to close its stomata. Symptoms characteristic of excessive auxin are evident in the developing mutant. Among these symptoms are branch and leaf epinasty, excessive rooting along the stem, and increased apical dominance. By using a leucine-incorporation assay, spray of whole plants with 2,4-dichlorophenoxyacetic acid, and wheat coleoptile bioassay, indications were found of an excess of activity and concentration of auxin-like substances in shoots of young and mature mutant plants. The wheat coleoptile bioassay also revealed a much lower amount of substances with abscisic acid-like activity in the mutant compared with the normal plant. In contrast to the appearance during ontogeny of morphological symptoms characteristic of auxin excess in the mutant, the absolute amount of auxin-like substances and their activity in incorporation of leucine decreased with age. A parallel decrease of the concentration and activity of auxin-like compounds was also found in the normal plant. The concentration of abscisic acid-like substances increased with age in both genotypes. The disagreement between the increasing morphological symptoms and the decrease of auxin-like activity and concentration is discussed, together with the possibility of a causal relationship between auxin-and abscisic acid-like activity and stomatal behavior.     

Urea derivatives on the move: cytokinin-like activity and adventitious rooting enhancement depend on chemical structure

Urea derivatives are synthetic compounds, some of which have proved to be positive regulators of cell division and differentiation. N -phenyl- N '-(2-chloro-4-pyridyl)urea (forchlorofenuron, CPPU) and N -phenyl- N '-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ), well known urea cytokinin representatives, are extensively used in in vitro plant morphogenesis studies, as they show cytokinin-like activity often exceeding that of adenine compounds. In recent years, renewed interest in structure–activity relationship studies allowed identification of new urea cytokinins and other urea derivatives that specifically enhance adventitious root formation. In this review, we report the research history of urea derivatives, new insights into their biological activity, and recent progress on their mode of action.     

The Weak Cytokinins N,N′-bis-(1-naphthyl)urea and N,N′-bis-(2-naphthyl)urea may Enhance Rooting in Apple and Mung Bean

The present research investigates the possibility that 2 weak urea-type cytokinins, the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea, enhance adventitious root formation. The rooting activity was assessed using the stem slice test, the mung bean rooting test and the rooting of apple microcuttings. The two compounds influenced the adventitious rooting process differently as regards the bioassay used. In the stem slice test, in the presence of exogenous auxin, both compounds enhanced the rooted slice percentage. In mung bean shoots, the N,N′-bis-(1-naphthyl)urea enhanced the root formation at the lowest concentration used (0.01 μM) while the N,N′-bis-(2-naphthyl)urea enhanced rooting at higher concentrations. In the rooting test of apple microcuttings the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea slightly enhanced only the mean root number per microcutting.     

Adventitious rooting adjuvant activity of 1,3-di(benzo[d]oxazol-5-yl)urea and 1,3-di(benzo[d]oxazol-6-yl)urea: new insights and perspectives

Here we report new insights on the adventitious rooting adjuvant activity of 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU), both symmetrically substituted urea derivatives that do not show either auxin- or cytokinin-like activity per se. Our data demonstrate that these synthetic molecules enhance adventitious rooting in distantly-related herbaceous and woody species, in the presence of endogenous or exogenous auxin. For the first time, we report that BDPUs enhance adventitious rooting in the presence of either indole-3-butyric acid (IBA) or 1-naphtalene acetic acid and that their optimal concentration depends on the strength of the exogenous auxin. Trying to understand the mode of action of BDPUs, we also show that their adventitious rooting adjuvant activity correlates with high mRNA levels of auxin-responsive genes related to the adventitious rooting process at the very early stages of adventitious rooting, before the activation of cell divisions in pine hypocotyls cuttings. The high mRNA levels are measured in the presence of low auxin concentrations and BDPUs. The mRNA levels quantified in these conditions are similar to those measured in the presence of high auxin concentrations but in the absence of BDPUs. In addition, the spatial distribution of endogenous auxin is localized in globular-shaped structures of cell divisions located centrifugal to the resin canals, at the positions of adventitious root formation, in the presence of urea derivatives and IBA after 6 days of the root induction process.     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

Auxin-like Growth Activity of Phenylacetonitrile

Phenylacetonitrile (PAN) and phenylacetic acid (PAA) showedauxin-like activity by stimulating elongation of wheat-coleoptilesections. Their auxin-like properties were also tested on theelongation of sections cut from hypocotyls of cress and sugar-beetseedlings, from petioles of sugar-beet leaves, and from epicotylsof pea seedlings. They were also tested for their ability toinduce adventitious roots on hypocotyls of cress and sugar-beetseedlings and on pea epicotyls. PAN was generally more activethan PAA on cress, and PAA more active than PAN on sugar-beetand peas. Steam distillates of cress seedlings contained a neutralsubstance with auxin-like activity and similar chromatographicproperties to PAN.     

Growth-regulating activity of N-benzyl- and O-benzyl-containing compounds belonging to a new group of synthetic analogues of natural auxins

We studied the effect of benzylamine, benzyl alcohol, and their derivatives (constituting a new group of synthetic analogues of natural auxins) on rooting of leaf and stem cuttings, rhizogenesis and growth of barley plantlets and tomato seedlings, and tomato plant productivity. These compounds promoted rooting of leaf and stem bean cuttings, increased rhizogenic activity, and stimulated the development of root systems in barley and tomato seeds. The activity of the compounds studied was similar to that of standard substances (3-indoleacetic acid potassium salt and 2-naphthylacetic acid). The benzyl group attached to the oxygen or nitrogen atom was shown to be the smallest molecular structure which provided auxin activity of the compounds. Derivatives of benzyl alcohol containing the quaternary ammonium fragment possessed auxin and anti-gibberellin (retardant) properties. They were selected by chemical synthesis of low-molecular-weight bioregulators with desired properties (a combination of chemical fragments with complementary physiological activity in the molecule). Auxin and anti-gibberellin (retardant) activities produced a synergistic effect. Germination of seeds treated with these compounds was accompanied by a more significant increase in the weight and length of roots (compared to standard auxins). The rate of seedling establishment reached 100%. The development of fruits and accumulation of reserve nutrient substances were synchronized and accelerated after spraying vegetating plants with solutions of studied compounds. The synergistic effect underlay a significant increase in the amount and quality of the crop (e.g., tomatoes).     

Cytokinin receptors are required for normal development of auxin-transporting vascular tissues in the hypocotyl but not in adventitious roots

Plants alter the architecture of their root systems to adapt to the environment by modulating post-embryonic (lateral and adventitious) root formation and growth. To understand better the genetic basis of this regulation, we screened ethylmethane sulfonate-mutagenized lines of Arabidopsis thaliana for adventitious rooting mutants. One mutant showed retardation of the primary root growth, no production of lateral roots and enhanced formation of adventitious roots. Mapping and genetic complementation revealed that this mutant named wooden leg-3 (wol-3) was an allele of ARABIDOPSIS HISTIDINE KINASE 4 (AHK4), a locus known to encode a cytokinin receptor. Although the vascular system of the primary root and hypocotyl in the wol-3 mutant was aborted, that of the adventitious roots was normally developed. In the hypocotyl of the wol-3 mutant, auxin signals accumulated around the aborted vascular system. The application of auxin to primary roots induced lateral root formation in the wol-3 mutant. Transport of radiolabeled auxin from the top of the hypocotyl to the primary root was inhibited in wol-3. Although only a single amino acid alteration had occurred in AHK4, the root morphology in the wol-3 mutant was quite similar to that in the ahk2 ahk3 ahk4 triple mutant, which is a loss-of-function mutant of the three cytokinin receptors. This implies that the functional disturbance of AHK4 affects the function of the other receptors. Our results suggest that cytokinin receptors are necessary for the formation of auxin-transporting vascular tissues in the hypocotyl, but not in adventitious roots.     

Multiple factors influence adventitious rooting in carnation (<Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L.) stem cuttings

Efficient propagation of uniform starting material is a critical requirement for mass production of most ornamental plants, including carnation. For some elite cultivars, the production of young plantlets is limited by poor adventitious root formation from stem cuttings. We previously characterized the molecular signature during adventitious rooting in two carnation cultivars, 2101-02 MFR and 2003 R 8, which were selected because of their contrasting rooting performance. To determine additional factors that contribute to the differences observed in adventitious rooting during the commercial scaling-up of this species, we characterized rooting performance and endogenous hormone levels in stem cuttings of these two cultivars during one production season. We found that stem cutting production declined during the harvest season in a cultivar-dependent manner. In addition, the initiation of adventitious roots in the stem cutting base depended on its endogenous auxin and cytokinin levels at harvest time, while their subsequent growth and development was mainly influenced by the physiological status of the mother plant at harvest time and of the stem cutting during the rooting process.     

Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current season's growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.     

Growth-regulating activity of <Emphasis Type="Italic">N</Emphasis>-benzyl- and <Emphasis Type="Italic">O</Emphasis>-benzyl-containing compounds belonging to a new group of synthetic analogues of natural auxins

We studied the effect of benzylamine, benzyl alcohol, and their derivatives (constituting a new group of synthetic analogues of natural auxins) on rooting of leaf and stem cuttings, rhizogenesis and growth of barley plantlets and tomato seedlings, and tomato plant productivity. These compounds promoted rooting of leaf and stem bean cuttings, increased rhizogenic activity, and stimulated the development of root systems in barley and tomato seeds. The activity of the compounds studied was similar to that of standard substances (3-indoleacetic acid potassium salt and 2-naphthylacetic acid). The benzyl group attached to the oxygen or nitrogen atom was shown to be the smallest molecular structure which provided auxin activity of the compounds. Derivatives of benzyl alcohol containing the quaternary ammonium fragment possessed auxin and anti-gibberellin (retardant) properties. They were selected by chemical synthesis of low-molecular-weight bioregulators with desired properties (a combination of chemical fragments with complementary physiological activity in the molecule). Auxin and anti-gibberellin (retardant) activities produced a synergistic effect. Germination of seeds treated with these compounds was accompanied by a more significant increase in the weight and length of roots (compared to standard auxins). The rate of seedling establishment reached 100%. The development of fruits and accumulation of reserve nutrient substances were synchronized and accelerated after spraying vegetating plants with solutions of studied compounds. The synergistic effect underlay a significant increase in the amount and quality of the crop (e.g., tomatoes).Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 243–249.Original Russian Text Copyright © 2005 by Gafurov, Makhmutova.     

Cytokinin- and auxin-like activity in Cyanophyta and microalgae

Seven strains of axenic unicellular Chlorophyta and three strains ofaxenic Cyanophyta were selected for their high growth rate and positivecytokinin-like activity in the Cucumber Cotyledon Expansion Bioassay. Ethanolicextracts of these ten strains were purified using cation exchange resin andpaper chromatography and then screened for cytokinin-like activity using theSoybean Callus Bioassay. No trend was detected in the Cyanophyta with onespecies having two peaks of cytokinin-like activity co-eluting with zeatinderivatives and iso-pentenyladenine derivatives and theother two species having only one peak of activity co-eluting with zeatinderivatives. Four Chlorophyta only had one peak of cytokinin-like activity,co-eluting with zeatin derivatives, while two species had two peaks of activityco-eluting with zeatin and iso-pentenyladeninederivatives.One Chlorophyta species had no detectable cytokinin-like activity. The same tenstrains were also tested for auxin-like activity. The water extract of sevenstrains demonstrated auxin-like activity in the Cucumber Cotyledon RootFormation Bioassay. No auxin-like activity was detected in any of the strainsusing the Mung Bean Bioassay after being extracted in methanol and purified byphase partitioning with phosphate buffer and ethyl acetate.     

Regeneration of calabrian pine from juvenile needles

Cytokinins applied in an agar medium induced adventitious buds on cultured needles from seedling of Pinus brutia Ten. Cytokinins applied as pulses to the explants prior to culture were less effective. Irrespective of the mode of cytokinin application, 8 weeks was the time required to bring about bud formation. Organogenetic potential of the cultured needles decreased with chronological age of the explanted seedlings. The induced buds grew into elongated shoots on culture medium without cytokinins, but the inclusion of activated charcoal (1%) doubled the elongation rate. There was an indication that mixtures of cytokinins were more effective than separate cytokinins in producing buds on explants, but the difference between treatments did not achieve statistical significance. Parenchyma cells in mesophyll layers were evidently the target cells responding to the culture conditions. After a period of activity and division in these cells, meristematic zones developed which later led to formation of bud primordia, and subsequently these primordia developed into well-formed adventitious buds. Subsequent rooting (64%) of shoots was achieved using a combination of two auxins and a low level of cytokinin.     

The role of cytokinins in rooting of stem slices cut from apple microcuttings

ABSTRACT

We examined the role of cytokinins in rooting of 1-mm stem slices cut from microcuttings of the apple rootstock ‘Jork 9?s. Various types of cytokinins inhibited the rooting of apple stem slices to different extents. Highest inhibition was obtained with thidiazuron and benzylaminopurine. Remarkably, isopentenyladenine and isopentenyladenosine enhanced rooting at low concentration (at the optimal concentration of 0.1 μM by 53 and 19%, respectively). We also examined the effect of lovastatin and simvastatin. These drugs are putative cytokinin-synthesis inhibitors. Both inhibited rooting and inhibition was partially reversed by simultaneous addition of zeatin. Moreover, in the presence of lovastatin a higher concentration of zeatin had to be applied to achieve inhibition of rooting than in the absence of the drug. This data indicates that these compounds indeed inhibited cytokinin synthesis. One-day pulses with lovastatin strongly blocked rooting when given just after cutting the slices but had no effect after that. Adding zeatin simultaneously reversed inhibition completely. In conclusion, our data confirm that cytokinins may strongly inhibit rooting but they also show that at low concentration, certain cytokinins enhance rooting. Moreover, synthesis of cytokinin is essential during root formation. We hypothesise that cell division initiated by a relatively high endogenous level of cytokinins just after cutting the slices is a necessary, initial step in adventitious root formation.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

Ethylene as an endogenous inhibitor of root regeneration in tomato leaf discs cultured in vitro

We examined ethylene effects on root regeneration in tomato leaf discs cultured in vitro. Applied ethylene or Ethephon did not stimulate rooting in the leaf discs. In the presence of indoleacetic acid. 5 × 10^-6M, these substances significantly inhibited root formation. Ethylene production (nl C₂H₄· (24 h)^-1. flask^-1) was positively correlated with increased IAA concentrations at various times during the culture period and, as a consequence, with the rooting response after 168 h. However, separate testing of equimolar concentrations of seven different auxins and auxin-like compounds showed no positive correlation between the rate of ethylene production and subsequent rooting response. Aeration of gas-tight flasks containing leaf discs and absorption of ethylene evolved from the discs by mercuric perchlorate in gas-tight flasks or pre-treatment of leaf discs with AgNO₃ significantly enhanced IAA induced root regeneration. Thus, these studies indicate that ethylene is not a rooting hormone per se. Furthermore, ethylene (whether applied externally or synthesized by the tissue) does not appear to account for the ability of auxin to stimulate rooting.