Cytokinin-like activity of N,N%

Plant Science 160 (2001) 1055– 1065
www.elsevier.com/locate/plantsci
Cytokinin-like activity of N,N%-diphenylureas.
N,N%-bis-(2,3-methylenedioxyphenyl)urea and
N,N%-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious root
formation in apple rootstock M26 (Malus pumila Mill.)
Ada Ricci a,*, Angela Carra a, Anna Torelli a, Cesare Augusto Maggiali b,
Giovanni Morini b, Camillo Branca a
a
Dipartimento di Biologia E6oluti6a e Funzionale, Via delle Scienze 11 /A, I-43100 Parma, Italy
b
Dipartimento Farmaceutico, Via delle Scienze 7, I-43100 Parma, Italy
Received 18 July 2000; received in revised form 23 January 2001; accepted 29 January 2001
Abstract
Vegetative propagation of cuttings is a widespread method to multiplicate plants. Adventitious root formation is a key step in
vegetative propagation and considerable progress has recently been made in understanding root formation. But, in spite of the
efforts made, no new rooting treatments have been developed. Here, we report for the first time, that N,N%-bis-(2,3-methylenedioxyphenyl)urea and N,N%-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious root formation in microcuttings of Malus
pumila Mill. rootstock M26. Roots emerge without auxin supplementation in the darkness, transfer in hormone free medium, or
callus formation. With the use of different bioassays, we also demonstrate that these two diphenylurea derivatives do not show
cytokinin- or auxin-like activity. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Adventitious rooting; Auxin-like activity; Cytokinin-like activity; Diphenylurea derivatives; Malus pumila Mill.
1. Introduction
Several billion rooted plantlets transferred to
the soil per year are obtained by vegetative propagation of cuttings, whether by classical multiplication methods or, in many cases, by in vitro
micropropagation (MP). This equals a commercial
value of many billion dollars, 35–75% of which is
spent for adventitious root formation [1,2]. These
high costs are due to the steps necessary to induce
adventitious root formation, such as a 3–10 day
period of darkness in the presence of a hormonal
supplementation and a transfer to a hormone free
(HF) medium in the light [2,3], and to the losses
that can occur during the acclimatization phase.
* Corresponding author. Fax: +39-0521-905403.
E-mail address: cbteam@biol.unipr.it (A. Ricci).
Owing to the economic importance, many authors have tried to improve rooting and have
investigated the effects of any kind of plant
growth regulators [4] and the influence of non-hormonal compounds in vitro. Putrescine seems to be
involved in the inductive phase of the adventitious
rooting process, but it is unable per se to induce
adventitious root formation when supplemented at
physiological doses [5]. Phenolic compounds (i.e.
pyrogallol, catechol, phloroglucinol, ferulic acid)
are reported to act synergistically with auxin on
adventitious rhizogenesis [6,7]. Auderset et al. [8,9]
have shown that root formation of soybean and
apple cuttings is stimulated by dithiothreitol and
reduced glutathione in presence of auxin shock.
Despite the great amount of research, no rooting
substance leads to a practical application without
some kind of auxin supplementation.
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 3 5 9 - 4
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A. Ricci et al. / Plant Science 160 (2001) 1055–1065
It is a well-known fact that some phenyl- and
diphenylurea (DPU) derivatives show cytokininlike activity, such as a strong positive regulation
of cell division and shoot regeneration [10].
Among these kinds of compounds, the Nphenyl-N%-1,2,3-thiadiazol-5-ylurea (thidiazuron,
TDZ) which was developed for mechanized harvesting of cotton bolls, has proved to be a
highly efficacious bioregulant of morphogenesis
in the tissue culture of many plant species. In
our work on synthetic auxin- and cytokinin-like
compounds [11 – 14], we synthesized some DPU
derivatives (Fig. 1). They were tested as cytokinins on tomato regeneration test, Malus
pumila Mill. M26 rootstock cutting MP, chlorophyll level determination in cucumber cotyledons, Pg5-GUS gene expression in tobacco
protoplasts in the presence of auxin. Owing to
the unexpected root formation in M26 cutting
MP, a suitable root inducing test on M26 microcuttings was performed. The same compounds
were tested on a root inducing assay designed to
confirm their root inducing capacity using Lycopersicon aesculentum var. Alice seedlings. The
possible auxin-like activity was also tested on
pea stem elongation test, tomato cotyledon rooting test, Pg5-GUS gene expression in tobacco
protoplasts in the presence of cytokinin and apple stem slice rooting test.
2. Materials and methods
2.1. Chemicals
2.1.1. General notes
The compounds tested, all neosynthesized but
compound 4 [15], were recrystallized from EtOH,
EtOH – H2O, or 1,4-dioxane. The various reactions were checked by TLC on silica gel
(Kieselgel 60, F 254; Merck), with CHCl3 –EtOH
(95:5) or (98:2), as eluents. For Soxhlet extractions, calcium-type silica gel (NM Kieselgel 60,
0.06 – 0.2 mm) was used. Mps: uncorrected. Only
the most significant peaks of IR (KBr) are reported. 1H-NMR spectra (DMSO-d6) agree with
the structural formulae. The results of elemental
analysis (C, H, N) were within 90.3% of the
theoretical values.
2.1.2. Compound 4,
N,N%-bis-(3,4 -methylenedioxyphenyl)urea, and
3,4 -(methylenedioxy)aniline
According to the method of Neville et al. [16],
diphenylphosphoryl azide (17.4 g, 0.06 mol) and
triethylamine (6.50 g, 0.06 mol) were added in
one batch to a boiling solution of 3,4-methylenedioxybenzoic acid (piperonylic acid) (10 g, 0.06
mol) in 1,4-dioxane (150 ml) and ter-butyl alcohol (150 ml); the mixture were stirred under
reflux for a further 10 h, then concentrated in
vacuo. The residue was taken up in CHCl3 –CCl4
(1:1; 330 ml): an insoluble bright crystalline
white solid was isolated by filtration and identified as N,N%-bis-(3,4-methylenedioxyphenyl)urea,
compound 4 (3.40 g). The remaining filtrate was
washed with successive portions of 5% aqueous
citric acid (3×50 ml), water, saturated aqueous
sodium hydrogen carbonate, and finally with saturated brine; a further 1.3 g of compound 2
were recovered from the ‘‘interphase’’. The organic layer was dried (Na2SO4), silica gel (10 g)
was added and the solvents evaporated. The
residual powder was placed in a Soxhlet apparatus and exhaustively extracted (24 h) with etherlight petroleum (30:70) solvent mixture (500 ml).
Evaporation of the solvents gave a product (4 g;
crude
t-butyl-3,4-methylenedioxyphenylcarbamate) which was directly hydrolyzed (EtOH 90
ml, 5 N HCl 65 ml; 24 h stirring at room temperature). After evaporation of the ethanol solvent, the aqueous layer was washed with ether
(3×45 ml) and to the resulting acid solution,
cooled in an ice-bath, solid potassium hydroxide
was added, to pH 7; the filtrate extracted with
ether (3×70 ml) and the organic layers dried.
The solvent was evaporated to leave 3,4(methylenedioxy)aniline, a yellow viscous oil (1.8
g), which was used as such in the next reaction
(compound 1). Likewise, compounds 2, 6, 8 and
the respective amines were prepared.
2.1.3. Compounds 1,3,5 and 7
The appropriate amine (0.0085 mol) was dissolved in benzene (30 ml) and to the boiling
solution phenylisocianate (1.12 g, 0.0095 mol) in
one batch was added. The mixture was stirred
under reflux for 1 h, then filtrated and the
whitish solid recrystallized. Yield 60%.
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
Fig. 1. Scheme of synthesis of the DPU derivatives.
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1058
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
2.1.4. Analytical data
Compound 1: N-(2,3-methylenedioxyphenyl)-N%phenylurea, m.p. 195 –196°C; IR (cm − 1) 3300,
1630; 1H-NMR(d) 8.94 (1H, s, NH); 8.32 (1H, s,
NH); 7.54 (1H, d, Ph); 7.43 (2H, dd, Ph); 7.26 (2H,
t, Ph); 6.98-6.93 (1H, m, Ph); 6.77 (1H, t, Ph); 6.59
(1H, dd, Ph); 6.04 (2H, s, OCH2O).
Compound 2: N,N%-bis-(2,3-methylenedioxyphenyl)urea, m.p. 293 –294°C; IR (cm − 1) 3280, 1650;
1
H-NMR(d) 8.74 (2H, s, NH); 7.54 (2H, d, Ph); 6.76
(2H, t, Ph); 6.04 (4H, s, OCH2O).
Compound 3: N-(3,4-methylenedioxyphenyl)-N%phenylurea, m.p. 204 –205°C; IR (cm − 1) 3280,
1640; 1H-NMR(d) 8.54 (1H, s, NH); 8.50 (1H, s,
NH); 7.42 (2H, dd, Ph); 7.28-7.19 (3H, m, Ph); 6.95
(1H, t, Ph); 6.83-6.73 (2H, m, Ph); 5.96 (2H, s,
OCH2O).
Compound 4: N,N%-bis-(3,4-methylenedioxyphenyl)urea, m.p. 275 –277°C; IR (cm − 1) 3280, 1620;
1
H-NMR(d) 8.43 (2H, s, NH); 7.16 (2H, d, Ph);
6.82-6.71 (4H, m, Ph); 5.95 (4H, s, OCH2O).
Compound 5: N-(2,3-dimethoxyphenyl)-N%-phenylurea, m.p. 142 –143°C; IR (cm − 1) 3320, 1650;
1
H-NMR(d) 9.29 (1H, s, NH); 8.33 (1H, s, NH);
7.81 (1H, dd, Ph); 7.45 (2H, d, Ph); 7.28 (2H, t, Ph);
6.97 (2H, m, Ph); 6.67 (1H, dd, Ph); 3.80 (3H, s,
OCH3); 3.76 (3H, s, OCH3).
Compound 6: N,N%-bis-(2,3-dimethoxyphenyl)urea, m.p. 178 – 179°C; IR (cm − 1) 3280, 1640;
1
H-NMR(d) 9.05 (2H, s, NH); 7.82 (2H, d, Ph); 6.97
(2H, t, Ph); 6.67 (2H, d, Ph); 3.79 (6H, s, OCH3);
3.75 (6H, s, OCH3).
Compound 7: N-(3,4-dimethoxyphenyl)-N%-phenylurea, m.p. 180 –182°C; IR (cm − 1) 3280, 1620;
1
H-NMR(d) 8.53 (1H, s, NH); 8.45 (1H, s, NH);
7.43 (2H, dd, Ph); 7.28-7.23 (2H, m, Ph); 7.19 (1H,
s, Ph); 6.97-6.86 (3H, m, Ph); 3.73 (3H, s, OCH3);
3.70 (3H, s, OCH3).
Compound 8: N,N%-bis-(3,4-dimethoxyphenyl)urea, m.p. 209 – 210°C; IR (cm − 1) 3300, 1630;
1
H-NMR(d) 8.36 (2H, s, NH); 7.17 (2H, s, Ph); 6.85
(4H, m, Ph); 3.73 (6H, s, OCH3); 3.70 (6H, s,
OCH3).
All the DPU derivatives were dissolved in
dimethylsulfoxide (DMSO) and the final concentration of DMSO in the medium did not exceed 0.2%
[16].
2.2. Tomato regeneration test
Twelve cotyledon explants, obtained from
seedlings cultivated in vitro, were plated on MS
medium [17] plus the DPU derivatives, as putative
cytokinins at 1, 5 and 10 mM, both alone and in the
presence of 20 mM 1,2-benzisoxazole-3-acetic acid
(BOAA), as auxin [18]. The results were compared
with that TDZ in the same culture conditions. After
2 week incubation, the cotyledon explants were
transferred to a hormone free medium (HF) and the
percentage of regenerated shoots was checked 2
weeks later. The experiments were carried out in
triplicate, and repeated three times.
2.3. Micropropagation of apple cuttings
Microcuttings of M. pumila Mill. rootstock M26
were obtained from Dr. E. Caboni (Istituto Sperimentale per la Frutticoltura, Roma, Italy). The
cuttings deprived of apices were propagated on a
MP medium (MS salts plus 0.4 mg l − 1 thiamine
HCl, 0.5 mg l − 1 nicotinic acid, 0.5 mg l − 1 pyridoxine HCl, 100 mg l − 1 myo-inositol, 10 g l − 1 sucrose,
20 g l − 1 sorbitol, 0.65% agar B&V, 5.8 pH). The
MP medium was supplemented with 1.3 mM benzylaminopurine (BAP), as control, or 1.3 mM DPU
derivatives, as cytokinins, and 0.25 mM indole-3-butyric acid (IBA). Incubation was at 26°C at a light
intensity of 27 mmol m − 2 s − 1 under 16 h photoperiod. After 3 week culture the microcuttings were
checked.
2.4. Chlorophyll le6el determination
Chlorophyll levels were determined as previously
described [19] with minor modifications. Cucumber
seeds (Cucumis sati6us L.) were soaked for 4 h in
running tap water and germinated on moist filter
paper in the darkness at 26°C for 1 week. Expanded
cotyledons were excised in dim green light and
floated in 5 cm Petri dishes containing 5 ml of DPU
derivative solutions at three different concentrations: 0.1, 1 and 10 mM. Each plate contained 10
cotyledons with the adaxial face down. Control was
performed with water and the same concentrations
of TDZ.
After 17 h incubation in the darkness at 26°C, the
dishes were moved to a light intensity of 27 mmol
m − 2 s − 1 for 3 h at 26°C. Chlorophyll was then
extracted from weighed cotyledons with 80% acetone, and the chlorophyll concentration was calculated as described [20]. Experiments were carried
out in triplicate, and repeated three times.
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
2.5. Bioassay for Pg5 -GUS gene expression
The bioassay for Pg5-GUS gene expression is
based on the expression of the chimeric Pg5-GUS
gene in protoplasts of Nicotiana tabacum transformed plants [21]. This chimeric gene, consisting
of A. tumefaciens T-DNA gene 5 promoter (Pg5)
fused to the b-glucuronidase coding sequence
(GUS), is slightly induced by cytokinin or auxin
alone, but is strongly activated when both growth
regulators are added to the culture medium. We
exploited this fact and tested compounds 2 and 4
separately either in the presence of cytokinin, to
check their auxin nature, or in the presence of
auxin, to check their cytokinin nature. Seeds of
Pg5-GUS transformed N. tabacum cv. Petit Havana (SR1) were a gift from W. Boerjan (Universiteit Gent, Belgium). Seed sterilization, in vitro
plant growth and tobacco protoplast preparation
were carried out as previously described [22]. An
average of 5× 106 protoplasts were resuspended in
2 ml of K3 medium [22] containing 0.4 M glucose.
Culture conditions were as follows:
“ hormone free medium;
“ 1 mM naphthalene-acetic acid (NAA) or 1 mM
zeatin riboside (ZR);
“ 1 mM NAA plus the following concentrations
of the DPU derivatives: 0.02, 0.2, 2 and 20 nM,
to test their cytokinin-like activity;
“ 1 mM NAA plus the same concentrations of
TDZ (0.02, 0.2, 2 and 20 nM) used as control;
“ 1 mM ZR plus the following concentrations of
the DPU derivatives: 1, 10 and 100 nM, to test
their auxin-like activity; and
“ 1 mM ZR plus the same concentrations of NAA
(1, 10 and 100 nM) used as control.
The rest of the experimental procedure was
performed according to Ricci et al. [22]. Experiments were carried out in duplicate, and repeated
twice.
2.6. Rooting of apple cuttings
M. pumila Mill. rootstock M26 microcuttings, 2 – 2.5 cm in length excised from 3-week
old cultures, were incubated in MP medium supplemented with 1, 5, 10 and 15 mM of N,N%-bis(2,3-methylenedioxyphenyl)urea (compound 2) or
N,N%-bis-(3,4-methylenedioxyphenyl)urea
(compound 4) under the following culture conditions:
1059
at light intensity of 27 mmol m − 2 s − 1 under 16
h photoperiod;
“ for 6 days in the darkness, then at light intensity of 27 mmol m − 2 s − 1 under 16 h photoperiod; and
“ for 6 days in the darkness, then transferred in
HF medium at light intensity of 27 mmol m − 2
s − 1 under 16 h photoperiod.
For each culture condition, controls were performed with microcuttings cultured in HF medium
and in the presence of the same concentrations of
IBA. In all the experiments the roots were counted
after 2 weeks. For each experiment 50 microcuttings were used.
“
2.7. Rooting of tomato seedlings
Tomato seeds (L. aesculentum var. Alice) were
sown and germinated under sterile conditions in
half strength MS medium without sucrose, 0.8%
agar, 5.8 pH. Incubation was at 26°C and 27 mmol
m − 2 s − 1 light intensity under 16 h photoperiod.
One-week old seedlings were divided into two
groups: in one, the seedlings were deprived of their
roots; and in the other, the seedlings were deprived
of their cotyledons too. The two groups were then
treated in the same way: the seedlings were plated
in MS medium containing 30 g l − 1 sucrose and
0.8% agar, 5.8 pH. Four different concentrations
of compounds 2 or 4 were used: 0.1, 0.5, 1 and 5
mM. HF medium and the same concentrations of
IBA were used as controls. The number of roots
was counted after 10 days. For each experiment 50
seedlings were used.
2.8. Pea stem elongation test
The activity of the compounds 2 and 4 on cell
elongation was tested on etiolated pea stem segments (Pisum sati6um var. Alaska) measuring their
increase in fresh weight [24]. The compounds were
tested at four different concentrations: 0.1, 1, 10
and 100 mM. Water and the same concentrations
of indole-3-acetic acid (IAA) were used as controls. The experiments were carried out in triplicate, and repeated twice.
2.9. Rooting of tomato cotyledons
Tomato cotyledons, var. Alice, were obtained
and cultivated in vitro as previously described [18].
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
1060
Ten cotyledon explants were plated on MS
medium plus compounds 2 and 4 separately, as
auxins, at 0.5, 1 and 5 mM. HF medium and the
same concentrations of IBA were used as controls.
The number of roots was counted after 10 days.
For each experiment 50 samples were used.
2.10. Rooting of apple stem slices
The experiment was performed according to
Van der Krieken et al. [23]. M. pumila Mill. rootstock M26 stem slices having :1 mm thickness
were cut with a razor blade from shoots of 1.5 –2
cm obtained with the MP method described
above. Groups of 25 slices were cultured in Petri
Table 1
Effect of DPU derivatives on shoot regeneration at three
different concentrations (1, 5 and 10 mM) in the tomato
cotyledon bioassay, in the absence (−) and presence (+) of
20 mM BOAA as auxina
Concentration (mM)
Explants forming shoots (%)
1
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
TDZ
a
1
2
3
4
5
6
7
8
5
−
+
−
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4.3 0
0
0
0
0
43
59
10
+
−
8.5 0
0
0
0
0
0
0
5.6 0
4.5 0
0
0
0
0
83
68
The cytokinin-like activity of all the DPU
derivatives was tested on the following assays.
+
2.6
0
0
0
4.8
0
0
0
92
Table 2
Effect of DPU derivatives on apple cutting MP at 1.3 mMa
All the DPU derivatives are unable to induce
shoot regeneration at all the concentrations tested,
both alone and in the presence of 20 mM BOAA,
as auxin (Table 1).
3.2. Micropropagation of apple cuttings
All the DPU derivatives are unable to induce
the formation of new microcuttings in the MP of
M. pumila Mill. rootstock M26 (Table 2) but,
surprisingly, compounds 2 and 4 induced adventitious roots.
3.3. Chlorophyll le6el determination
Mean shoot number
Concentration (mM)
1.3
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
BAP
0
0
0
0
0
0
0
0
12
a
3. Results
3.1. Tomato regeneration test
The same concentrations of TDZ were used as control.
1
2
3
4
5
6
7
8
dishes with the apical side on a nylon mesh put on
MP medium supplemented with compounds 2 and
4 separately, as auxin at 1, 3.2, 5 and 10 mM. The
dishes were incubated upside down at light intensity of 27 mmol m − 2 s − 1 at 26°C under 16 h
photoperiod. Control was performed with apple
stem slices cultured in HF medium and the same
concentrations of IBA.
In another set of experiments, apple stem slices
were incubated in the darkness under the same
conditions described above. After 6 days of darkness the slices were transferred to HF medium and
incubated at light intensity of 27 mmol m − 2 s − 1 at
26°C under 16 h photoperiod [23]. The number of
roots was counted after 14 days. The experiments
were carried out in triplicate, and repeated twice.
The same concentration of BAP was used as control.
All the DPU derivatives are unable to increase
chlorophyll level at all the concentrations tested
(Table 3).
3.4. Bioassay for Pg5 -GUS gene expression (as
cytokinins)
All the DPU derivatives are unable to induce
Pg5-GUS activity, when tested as cytokinins in the
presence of 1 mM NAA. Fig. 2(A) shows only the
relative graphs for compounds 2 and 4.
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
Table 3
Effect of DPU derivatives on chlorophyll level at three different concentrations (0.1, 1 and 10 mM) using the cucumber
cotyledon bioassaya
Concentration (mM)
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
TDZ
1
2
3
4
5
6
7
8
Chlorophyll level of the control
(%; H2O)
0.1
1
10
−34.35
3.68
−23.92
−50.31
−18.41
−46.01
−34.35
7.97
6.13
−25.76
−3.06
−21.47
−49.07
−17.79
−29.44
−19.63
−34.96
8.58
−24.54
−17.17
−17.79
−37.42
−7.36
−23.31
−3.06
−29.44
11.65
1061
were observed when the microcuttings cultured in
the continuous presence of compounds 2 and 4
separately, were transferred to the light after 6
days in the darkness. When compounds 2 and 4
were separately tested at 5, 10 and 15 mM, the
a
The results are expressed as a percentage of the control
(H2O alone) by the formula (T−C/C)×100. The same concentrations of TDZ were used as control. The standard error
is less than 10% for each measurement (n= 9).
According to these results, no DPU derivative
shows cytokinin-like activity. However, during
the apple MP, N,N%-bis-(2,3-methylenedioxyphenyl)urea (compound 2) and N,N%-bis-(3,4methylenedioxyphenyl)urea (compound 4) induced
adventitious roots. Therefore, these two compounds were tested in the rooting bioassays described below.
3.5. Rooting of apple cuttings
Compounds 2 and 4 enhanced root formation in
microcuttings of M. pumila Mill. rootstock M26.
The best rooting stimulation was obtained when
the microcuttings were cultured in the light, in the
constant presence of 1 mM of compound either 2
or 4. In 70% and 91% of the microcuttings respectively (Table 4), the roots emerged directly from
the base of the microcuttings without callus formation. Under the same culture conditions, in the
presence of 1 mM IBA, the percentage of rooted
microcuttings was 75%, and only few roots
emerged from a large-sized callus. This percentage
went up to 86% when the microcuttings were
transferred to HF medium in the light, after a 6
day treatment with 1 mM IBA in the dark (best
auxinic rooting treatment). In this condition, several roots emerged from each microcutting, but a
callus formation was always present.
No significant differences in rooting percentages
Fig. 2. (A) Effect of TDZ, compounds 2 and 4 (as cytokinins)
on Pg5-GUS expression in the tobacco bioassay at four
different concentrations (0.01, 0.02, 0.2 and 2 nM) in the
presence of 1 mM NAA. The results express GUS activity as
pmol of 4-methyl-umbelliferone (MU)/mg protein/min. The
standard error is less than 10% for each measurement (n=4).
(B) Effect of NAA, compounds 2 and 4 (as auxins) on
Pg5-GUS expression in the tobacco bioassay at three different
concentrations (1, 10 and 100 nM) in the presence of 1 mM
ZR. The results express GUS activity as pmol of 4-methylumbelliferone (MU)/mg protein/min. The standard error is
less than 10% for each measurement (n= 4).
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
1062
Table 4
Effect of compounds 2 and 4 on adventitious rooting of apple microcuttings at 1 mMa
Concentration (mM)
Rooted cuttings (%)
0
Compound 2
Compound 4
IBA
HF
1
0
70.4
91.1
75.2
28.5
1
0
1
68.3
87.5
79.3
43.7
50.1
86.5
18.7
18.7
a
L – the microcuttings, cultured in the continuous presence of the compounds, in the light; D–L – the microcuttings are
cultured in the continuous presence of the compounds, are transferred to the light after 6 days in the darkness; D–L (HF) – after
6 day culture in the darkness in the presence of the compounds, the microcuttings are transferred to HF medium in the light. HF
and the same concentration of IBA were used as control. The results express the percentage of rooted microcuttings. The standard
error is less than 10% for each measurement (n= 50).
rooting percentage was not significantly different
from that of the HF condition. With the increasing
concentrations of IBA the number of rooted microcuttings diminished in all the culture conditions
tested.
3.6. Rooting of tomato seedlings
When the tomato seedlings were only deprived of
their root system, compounds 2 and 4 were able to
increase the number of adventitious roots. At 5 mM
the mean root number was similar to that obtained
with the same concentration of IBA (Table 5).
When the seedlings were deprived both of their root
system and their cotyledons, only IBA at 5 mM was
able to increase root formation (Table 6).
According to the results obtained with the rooting bioassays, the possible auxin-like activity of
compounds 2 and 4 was tested.
3.7. Bioassay for Pg5 -GUS gene expression (as
auxins)
Compounds 2 and 4 are unable to induce Pg5GUS activity, when tested as auxins in the presence
of 1 mM ZR (Fig. 2(B)).
stem at all the concentrations tested (Table 8). The
increase in fresh weight is not significantly different
from that obtained with H2O, used as control.
Table 5
Effect of compounds 2 and 4 on adventitious rooting of
tomato seedlings at 0.1, 0.5, 1 and 5 mMa,*
Concentration (mM)
0
Compound 2
Compound 4
IBA
HF
Compounds 2 and 4 are unable to induce cell
elongation on the third internode of etiolated pea
0.5
1
5
7.6
9
7.7
8.5
10.4
7.3
9
9
7.3
10.3*
11.7*
10.8*
6.6
HF and the same concentrations of IBA were used as
control. The results express the mean root number obtained
per seedling. The standard error is less than 10% for each
measurement (n= 50).
* Significantly different at 95% (F test).
Table 6
Effect of compounds 2 and 4 on adventitious rooting of
tomato seedlings deprived of cotyledons at 0.1, 0.5, 1 and 5
mMa,*
Concentration (mM)
Mean root number
0
Compound 2
Compound 4
IBA
HF
a
3.9. Pea stem elongation test
0.1
a
3.8. Rooting of apple stem slices
Compounds 2 and 4 are unable to induce roots
in apple stem slices at all the concentrations tested
(Table 7).
Mean root number
0.1
0.5
1
5
4.1
3.8
3.9
4.1
4
3.4
4
3.7
4.2
3.7
3.2
6*
3.9
HF and the same concentrations of IBA were used as
control. The results express the mean root number obtained
per seedling. The standard error is less than 10% for each
measurement (n= 50).
* Significantly different at 95% (F test).
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
1063
Table 7
Effect of compounds 2 and 4 on root formation using the apple stem slice bioassay at four different concentrations (1, 3.2, 5 and
10 mM)a
Concentration (mM)
Mean root number per slice
1
L
Compound 2
Compound 4
IBA
HF
0
D
3.2
5
10
L
D
L
D
L
D
L
D
0
0
0.5
0
0
0.7
0
0
1
0
0
2.3
0
0
1
0
0
0.7
0
0
0.7
0
0
0.9
0
a
Control was performed with the same concentrations of IBA. L: the apple stem slices are cultured in continuous light; D: after
6 days in the darkness, the apple stem slices are transferred to HF medium in the light. The standard error is less than 10% for
each measurement (n=6).
3.10. Rooting of tomato cotyledons
Compounds 2 and 4 are unable to affect root
formation in tomato cotyledon explants at all the
concentrations tested (Table 9). The number of
roots is not significantly different from that obtained in HF condition.
4. Discussion
None of our neosynthesized N,N%-diphenylurea
derivatives showed cytokinin-like activity. This is
consistent with the knowledge that substituents of
N,N%-diphenylurea phenyl rings with great steric
hindrance lower the cytokinin-like activity of the
compounds [10].
When we tried to micropropagate microcuttings
of M. pumila Mill. rootstock M26, surprisingly
compound 2 (N,N%-bis-(2,3-methylenedioxyphenyl)urea) and compound 4 (N,N%-bis-(3,4-methylenedioxyphenyl)urea) enhanced adventitious root
formation. Even this particular activity is related
to specific molecular structures. Our asymmetrical
compounds are actually unable to enhance adventitious root formation. But symmetrical molecular
structure is not the only requirement to be met in
order to obtain a greater number of adventitious
roots. In fact N,N%-bis-(2,3-dimethoxyphenyl)urea
and N,N%-bis-(3,4-dimethoxyphenyl)urea, i.e. compounds 6 and 8, respectively, are unable to enhance adventitious root formation. Besides,
neither N-(2,3-methylenedioxyphenyl)-N%-phenylurea nor N-(3,4-methylenedioxyphenyl)-N%-phenylurea, i.e. compound 1 and 3 respectively, are
able to increase the number of adventitious roots,
as only one methylenedioxy benzo-condensed
group is linked to urea matrix.
To verify if compounds 2 and 4 could be responsible per se for this activity, apple cuttings
were cultured with different doses of these two
DPU derivatives alone in the light. The results
were definitely surprising. When tested at 1 mM,
compounds 2 and 4 allowed root formation in
were separately tested at 5, 10 and 15 mM, the 70%
and 91% of the cuttings, respectively, as shown in
Table 4. Roots emerged after 15 days with no
auxin supplementation, no darkness treatment,
no transfer to HF medium in the light and no
callus formation (Fig. 3). Under the same cultureconditions, 1 mM IBA allowed root formation
in 75% of the cuttings, in which only few
roots emerged from a large-sized callus. This is
Table 8
Effect of compounds 2 and 4 on cell elongation measured as
the increase in fresh weight of the third internode of etiolated
pea stem, at four different concentrations (0.1, 1, 10 and 100
mM)a
Concentration (mM)
Compound 2
Compound 4
IAA
H2O
a
Increase of fresh weight (%)
0.1
1
10
100
15
11
39*
19
14
48*
15
14
54*
18
18
54*
11
Water and the same concentrations of IAA were used as
control. The standard error is less than 10% for each measurement (n=6).
* Significantly different at 95% (F test).
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
1064
Table 9
Effect of compounds 2 and 4 on root induction at three different concentrations (0.5, 1 and 5 mM) using the tomato cotyledon
explant bioassaya,*
Concentration (mM)
Mean root number
0.5
Compound 2
Compound 4
IBA
HF
2.2
6.1
7.1
5.1
1
90.6
91.2
90.9
91.2
4.1
6.9
6.7
5.1
5
91.5
90.9
91.3
91.2
2.2
6.3
14.9*
5.1
90.6
90.9
91.3
91.2
a
HF and the same concentrations of IBA were used as control. The standard error is less than 10% for each measurement
(n=50).
* Significantly different at 95% (F test).
Fig. 3. Example of rooted microcuttings obtained under the best culture conditions. 1 mM IBA – after 6 days of IBA treatment
in the darkness, the microcuttings were transferred to HF medium in the light (27 mmol m − 2 s − 1 under 16 h photoperiod); 1 mM
compound 2 – the microcuttings are cultured in the continuous presence of 1 mM compound 2 in the light (27 mmol m − 2 s − 1
under 16 h photoperiod); 1 mM compound 4 – the microcuttings are cultured in the continuous presence of 1 mM compound 4
in the light (27 mmol m − 2 s − 1 under 16 h photoperiod). Control was performed with microcuttings cultured in HF medium.
Picture was taken after 15 days of culture.
consistent with the knowledge that after 96 h of
treatment, auxin is no longer required and becomes inhibitory during the rooting differentiation
phase [2]. In fact, the rooting percentage increases
(86%) when the IBA period treatment was
reduced.
As compounds 2 and 4 were able to enhance
root formation also in the tomato seedlings deprived of their own roots, we performed different
bioassays to verify their possible auxin-like activity. The results show that the N,N%-bis-(2,3methylenedioxyphenyl)urea
and
the
N,N%-bis-(3,4-methylenedioxyphenyl)urea do not
behave as auxins.
In conclusion, our data demonstrate that
1. the rooting activity of N,N%-bis-(2,3-methylenedioxyphenyl)urea and N,N%-bis-(3,4-methylenedioxyphenyl)urea is strictly related to the
symmetrical presence of methylenedioxy
benzo-condensed group on urea matrix;
2. they might be able to cooperate with molecules
originating from other parts of the plants, as,
when the two compounds were tested on apple
stem slices or tomato seedlings deprived of
roots and cotyledons, root induction was
absent;
3. the N,N%-bis-(2,3-methylenedioxyphenyl)urea
and the N,N%-bis-(3,4-methylenedioxyphenyl)
urea do not show inhibitory effect on root
formation, as the best rooting condition is
A. Ricci et al. / Plant Science 160 (2001) 1055–1065
obtained when the apple microcuttings are left
in continuous contact with them (Table 4); and
4. they are the first non-hormonal compounds
showing a direct rhizogenetic activity. They
could be the starting point of a new category of
specific compounds favouring root formation,
that could cut down the adventitious rooting
costs in horticultural, agricultural and forestry
vegetative multiplication.
Work is in progress to elucidate the mechanism
by which the N,N%-bis-(methylenedioxyphenyl)
ureas might enhance rooting.
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