Ada Ricci

Auxin structure and activity on tomato morphogenesis in vitro and pea stem elongation

In order to understand better the relationship between auxin structure and activity on morphogenesis and cell elongation, six different auxins were tested on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. The auxins were: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1, 2-benzisoxazole-3-acetic acid (BOA), 1,2-benzisothiazole-3-acetic acid (BIA), 1-naphthalenacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). All these compounds obey the minimum requirement rules for auxin activity and all were effective on cell elongation. At the dose of 10 μM and in the absence of cytokinin, they all, except 2,4-D, induced roots, while in the presence of cytokinin they induced shoots, roots, hairy root-like filaments (HRLF) or callus depending on their concentration. The morphogenetic pattern did not change by varying cytokinin concentration. We conclude that auxin structure plays a minor role in morphogenesis or cell elongation, because it is only responsible for variations in the level of auxin activity.

Cytokinin-like activity of N'-substituted N-phenylureas

We have synthesized 14 N-phenylurea derivatives, differing in theheterocyclic portion linked in N′-position, and tested theircytokinin-like activity. Three different bioassays were used: the chlorophylllevel determination test, the bioassay for the expression of hormone-inducedchimeric Pg5-GUS gene and the tomato regeneration test, in which1,2-benzisoxazole-3-acetic acid (BOAA) was utilized as auxin. Thecytokinin-likeactivity showed by three of these compounds in the regeneration assay seems tobe related to their different heterocyclic nature. Results obtained indicatethat the N-phenyl-N′-1,3,4-thiadiazol-2-ylurea (compound 4), an isomer ofN-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ), in the absenceof auxin induces shoot regeneration in the 34,2% of the explantscultured; theN-phenyl-N′-(3-chloro-1,2-benzisothiazol-7-yl) urea (compound 10),structurally different from TDZ, in the absence of auxin induces shootregeneration in the 25,9% of explants, significantly lower than that ofTDZ (68,8%). N-phenyl-N′-benzothiazol-6-ylurea (compound 13),structurally different from TDZ, in the absence of auxin induces the99,5% of shoot regeneration, significantly different from that of theother substances. The addition of auxin in the cotyledon regeneration assayreduces the differences. The compound 13 could be considered a new phenylureaderivative with a highly specific 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.

In vitro micropropagation of the aquatic fern Marsilea quadrifolia L. and genetic stability assessment by RAPD markers

In order to conserve and multiply the aquatic fern Marsilea quadrifolia L., in a long-term in vitro procedure, the effects of different cytokinins, i.e., 6-benzylaminopurine, zeatine riboside, and N6-(2-isopentenyl)adenine, were investigated, varying their concentration and period of supplementation. No clear stimulatory effect on the de novo nodes produced per explant was detected when compared with hormone-free (HF) condition. On the contrary, the rhizome explant micropropagation was inhibited, the inhibition decreasing with the decreasing strength of cytokinins, though without reaching any significant enhancement. Since, as a consequence of the tissue culture procedure, the occurrence of somaclonal variation may introduce genomic alterations, genetic stability was assessed by random amplified polymorphic DNA (RAPD) analysis by comparing eight randomly selected micropropagated plants derived from repeated subcultures, with donor plant. Eighteen different primers generated 189 bands ranging from 100 to 3250 bp, and the same banding profiles were exhibited. No genomic alterations were evidenced in any of the micropropagated plants. Well-developed micropropagated plants were also successfully acclimatized under greenhouse condition. These positive results suggest that the in vitro HF micropropagation could be useful in the development of ex situ conservation programs of M. quadrifolia, even in order to possibly reintroduce the plants in their natural environment.

Visualizing the relevance of bacterial blue‐ and red‐light receptors during plant–pathogen interaction

The foliar pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) leads to consistent losses in tomato crops, urging to multiply investigations on the physiological bases for its infectiveness. As other P. syringae pathovars, Pst is equipped with photoreceptors for blue and red light, mimicking the photosensing ability of host plants. In this work we have investigated Pst strains lacking the genes for a blue-light sensing protein (PstLOV), for a bacteriophytochrome (PstBph1) or for heme-oxygenase-1. When grown in culturing medium, all deletion mutants presented a larger growth than wild-type (WT) Pst under all other light conditions, with the exception of blue light which, under our experimental conditions (photon fluence rate = 40 μmol m(-2) s(-1)), completely suppressed the growth of the deletion mutants. Each of the knockout mutants shows stronger virulence towards Arabidopsis thaliana than PstWT, as evidenced by macroscopic damages in the host tissues of infected leaves. Mutated bacteria were also identified in districts distant from the infection site using scanning electron microscopy. These results underscore the importance of Pst photoreceptors in responding to environmental light inputs and the partial protective role that they exert towards host plants during infection, diminishing virulence and invasiveness.

N,N′-bis-(2,3-methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea interact with auxin in enhancing root formation of M26 apple (Malus pumila Mill.) stem slices

Stem slices cut from micropropagated cuttings of apple rootstock M26 were cultured in the presence of indole-3-butyric acid (IBA) plus N,N′-bis-(2,3-methylenedioxyphenyl)urea or N,N′-bis-(3,4-methylenedioxyphenyl)urea, to verify if there was an interaction between them in enhancing root formation. The N,N′-bis-(methylenedioxyphenyl)ureas were supplemented after, before and in the simultaneous presence of auxin. Our data demonstrate that only the simultaneous presence of auxin and N,N′-bis-(methylenedioxyphenyl)ureas in the culture medium enhanced root formation on M26 stem slices. The percentage of rooted slices obtained in the presence of the mixtures was significantly different from that obtained in the presence of low auxin concentration alone (1µM). Moreover both the percentage of rooted slices and the number of roots per slice obtained in these culture conditions was not significantly different to that of the optimal auxinic treatment in which the auxin concentration was threefold higher.

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.

Weakly cytokinin-active diphenylurea derivatives influence adventitious rooting in M26 Malus pumila microcuttings

The cytokinin activity of 8 previously unstudied diphenylurea (DPU)derivatives, differing in either type or position of the substituents of thephenyl rings, was investigated. Cytokinin activity was assessed using thebetacyanin (so-called amaranthin) accumulation test and the tomato regenerationtest. We also assayed their capacity to enhance adventitious root formation inmicrocuttings of apple rootstock M26 and in a tomato cotyledon rooting test,while their possible auxin-like activity was tested using the pea stemelongation test. All the compounds showed weak cytokinin activity, and thismight be responsible for the enhanced adventitious rooting which is induced bysome of the compounds. None of the DPU derivatives showed auxin-like activity.

Effect of 1,2-benzisoxazole-3-acetic acid on plant regeneration from protoplasts of Nicotiana tabacum

SummaryBenzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.

Methoxylation modifies the activity of 1,2-benzisoxazole-3-acetic acid: 6,7-dimethoxy-1,2-benzisoxazole-3-acetic acid is an auxin antagonist in cytokinin mediated processes

Abstract The insertion of a methoxy group in different positions of the aromatic ring modifies the activity of 1,2-benzisoxazole-3-acetic acid (BOAA), a specific morphogenetic compound with no activity on cell elongation or root growth. Monomethoxylation in the 4- and 7-position is critical in determining the kind of activity: 4-OMeBOAA induces stem elongation, inhibits root growth and does not improve shoot production; 7-OMeBOAA inhibits stem elongation and shoot production and is unable to induce root growth. 6,7-OMeBOAA, inactive on stem elongation and root growth, is unable to induce the expression of Pg5-GUS gene in the presence of BAP and inhibits the expression of this gene when induced by BAP plus IAA. Furthermore, 6,7-OMeBOAA inhibits completely shoot production and can therefore be regarded as an auxin antagonist in these cytokinin-mediated processes.