Content uploaded by Arthur G Fett-Neto
Author content
All content in this area was uploaded by Arthur G Fett-Neto
Content may be subject to copyright.
-1
Role of auxin and its modulators in the adventitious rooting of Eucalyptus
species differing in recalcitrance
Cla
´
udia Martellet Fogac¸ a
1
and Arthur G. Fett-Neto
1,2,
*
1
Laborato
´
rio de Fisiologia Vegetal (Programa de Po
´
s-Graduac¸a
˜
o em Bota
ˆ
nica), Departamento de Bota
ˆ
nica,
UFRGS;
2
Programa de Po
´
s-Graduac¸a
˜
o em Biologia Celular e Molecular, Centro de Biotecnologia, Univer-
sidade Federal do Rio Grande do Sul (UFRGS), Caixa Postal 15005, Porto Alegre, RS, 91501–970 Brazil;
*Author for correspondence (e-mail: fettneto@cbiot.ufrgs.br; phone: +55-51-3167642; fax: +55-51-
33167309)
Received 8 March 2004; accepted in revised form 22 November 2004
Key words: Auxin, Eucalyptus, Light, Phenolics, Rooting, Silver
Abstract
It is well established that auxins play a central role in the determination of rooting capacity, which is
essential for vegetative propagation. Recent studies with apple trees have pointed to significant effects of
auxin stability, wound related phenolics and ethylene production in the control of adventitious rooting. In
the present study, a comparative analysis of the adventitious rooting of microcuttings of Eucalyptus saligna
(easy-to-root species) and Eucalyptus globulus (difficult-to-root species) was carried out with different types
of auxins, light intensities, presence or absence of apical meristem, different concentrations of phenolic
compounds and presence or absence of an ethylene action inhibitor. Parameters evaluated were the percent
rooting, number of roots per rooted cutting, length of longest root and mean rooting time. Results showed
that auxins of intermediate stability are more favorable to rooting (particularly for the recalcitrant species),
higher light intensities in the presence of exogenous auxins promote the rooting response, the absence of
meristematic apex or externally supplied phenolics are not limiting for the rooting induced by exogenous
auxins, and ethylene appears to play a minor role in the development of adventitious roots in microcuttings
of Eucalyptus, indicating that the rhizogenic response results from direct effect of auxins.
Introduction
Adventitious rhizogenesis in cuttings (ARC) is a
unique and complex process, essential in plant
propagation. Analyses of ARC of a number of
woody species, particularly using Malus as a
model, have led to the general recognition of three
phases in the process: (1) induction – defined as the
period in which no morphological events are
clearly observed, comprising the early molecular
and biochemical steps preceding morphological
modifications; (2) initiation – in which cell divi-
sions take place, root meristems are formed and
root primordia are established; (3) expression –
root growth and emergence out of cuttings occurs
(Kevers et al. 1997). The last two phases are often
referred to as the root formation phase.
Auxin plays a central role in the determination of
rooting capacity, and light conditions are known to
affect auxin metabolism and tissue sensitivity (Reid
et al. 1991). Auxin is biosynthesized from trypto-
phan, indole or indole glycerol phosphate and its
catabolism involves oxidative decarboxylation
by peroxidases (EC 1.11.1.7) or alternative
non-decarboxylating pathways. Auxins can also be
conjugated, usually with amino acids, sugars or
Plant Growth Regulation (2005) 45: 1–10 Springer 2005
DOI: 10.1007/s10725-004-6547-7
inositol, and become inactive; most conjugates are
resistant to oxidative enzymes and can revert to free
auxin, creating a useful mode of regulation of auxin
activity (Crozier et al. 2000). Auxin action involves
binding to a receptor protein and triggering of a
signal transduction cascade that probably involves
gene de-repression by proteolysis of transcriptional
regulators (AUX/IAA) via the ubiquitin-proteasome
pathway (Dharmasiri and Estelle 2004).
In adventitious rooting, higher auxin concen-
trations are required during the induction phase,
whereas during the formation phase the phyto-
hormone becomes inhibitory; this profile has been
observed in various plant species (De Klerk et al.
1999). Rooting responses are also strongly affected
by the endogenous auxin content and transport
rate. Shoot apexes are the main sources of
endogenous auxin. Stems have a characteristic
basipetal active transport of au xin through the
vascular parenchyma cells carried out by influx
and efflux transporters, namely AUX1 and PIN
proteins, respectively (Muday and DeLong 2001).
Most commercial propagation is done by root-
ing with indole butyric acid (IBA); other auxins
often used are indole acetic acid (IAA) and
naphthalene acetic acid (NAA). Auxin type effi-
cacy depends on the affinity for the auxin receptor
protein involved in rooting, on the concentration
of free auxin that reaches target competent cells,
on the amount of endogenous auxin, and on
metabolic stability (lower in IAA, intermediate in
IBA and higher in NAA) (De Klerk et al. 1999).
As shown for apple microcuttings, it may be
advantageous to use IAA to obtain root induction
and formation in a single culture medium (De
Klerk et al. 1999). In a number of species, perox-
idases have been shown to display a pattern of
activity during rooting that is typically minimum
at the inductive phase and maximum at the initi-
ation phase (e.g. Gaspar and Thorpe 1977; Mon-
cousin et al. 1988; Fett-Neto et al. 1992). The
auxin resistant, non-rooting mutant of tobacco,
rac displays higher peroxidase activity (both basic
and acidic), higher ethylene production and higher
lignification (Faivre-Rampant et al. 1998).
Monophenolics act generally as promoters of
peroxidase activity, whereas di and polyphenolics
act as peroxidase inhibitors (Lee et al. 1982). More
recently, phenolics (e.g. ferulic acid and
phloroglucinol) have been regarded as important
adjuvants during the first stages of the adventitious
rooting process in apple (along with other wound
related compounds, such as jasmonate), poten-
tially enhancing the competence of target tissues to
rooting; phenolics apparently act as antioxidants,
protecting IAA from oxidation and plant tissue
from oxidative stress due to wounding (De Klerk
et al. 1999). Flavonoids could modulate auxin
transport in vivo (Curir et al. 1990; Brown et al.
2001).
Ethylene is induced by high auxin concentra-
tions through the promotion of transcription of
1-aminocyclopropane-1-carboxylic acid (ACC)
synthase gene and may affect rooting responses
(Brock and Kauffman 1991). Ethylene may also
enhance the sensitivity to auxins (Visser et al.
1996). In apple, ACC promoted rooting in well
aerated systems such as leaf disks, but was inhib-
itory in agar-grown cuttings, presumably due to
toxic amounts of ethylene accumulated around the
basal stem (De Klerk et al. 1999). Ethylene induces
acidic peroxidases involved in lignin biosynthesis
and cellulases and pectinases that facilitate root
emergence through stem tissues (Gonza
´
lez et al.
1991; Faivre-Rampant et al. 1998). Ethylene may
also promote rooting by stimulating cytokinin
catabolism (Bollmark and Eliasson 1990).
The present study examined the roles of auxin
type and stability, irradiance level, phenolic
compounds, presence or absence of apex, and
presence or absence of an ethylene action
inhibitor in the main phases of adventi tious
rooting in Eucalyptus globulus (difficult-to-root)
and E. saligna (easy-to-root) microcuttings. The
objectives of the investigation wer e to identify
auxin-related factors conditioning rooting recal-
citrance, develop improved rooting protocols,
and to establish the importance of critical
treatments for the rooting of apple cuttings, a
model species for in vitro adventitious rooting of
woody plants, in Eucalyptus species.
Materials and methods
Plant material
Seeds of Eucalyptus globulus Labill and E. saligna
Smith were a gift from the cellulose company
Aracruz S.A. (Guaı
´
ba, RS, Brazil). Prior to the
experiments, seeds were was hed in distilled water,
immersed in 70% ethanol for 1 min, and immersed
2
in 1.5% NaClO with a few drops of detergent for
15 min. After four washes in sterile distilled water,
seeds were placed in 50 ml of germination nutrient
medium (Fett-Neto et al. 2001) in 500 ml glass
flasks; approximately 10 seeds were placed in each
flask. The photoperiod was 16 h of light (P.A.R. of
35 lmol m
2
s
1
at explant level), provided by
white fluorescent lamps; temperature was
27 ± 2 C. Three months after inoculation, 3 cm
long tip microcuttings wer e obtained from the
plantlets.
Culture conditions
Rooting assays followed the basic protocol de-
scribed in (Fett-Neto et al. 2001), involving a 4 day
induction passage in 10 mg l
1
IBA (Riedel de
Ha
¨
en, Germany), followed by transfer to au xin-free
formation medium containing 1 g l
1
activated
charcoal. All sterilization procedures, vial size and
medium preparation were as previously described
(Fett-Neto et al. 2001), except for explant density,
kept at two per vial.
To evaluate the role of auxin type in rooting, the
IBA in the induction medium was replaced with one
of the following auxins: naphthaleneacetic acid
(NAA, Carolina Biological, USA), indole acetic
acid (IAA, Sigma, USA), or indole acetic acid
aspartate (IAAasp, Research Organics, USA), all at
49.3 lM (equimolar with the auxin concentration in
the usual induction medium, using IBA). Auxin free
controls were also included in the experiments. All
auxins were added prior to autoclaving.
The effect of different light intensities (dark, 30,
and 100 lmol m
2
s
1
, provided by white fluores-
cent lamps) during both phases of rhizogenesis in
microcuttings was analyzed with and without
10 mg l
1
IBA exposure. The role of the meriste-
matic apex in the rooting response induced by
10 mg l
1
IBA was studied in microcuttings in
which the terminal apex was removed with a scalpel
prior to cutting inoculation. Cuttings derived from
the same position in the donor plants to avoid top-
ophysis effects.
In order to evaluate the role of phenolic com-
pounds in the rhizogenic response, various modi-
fications were made to the basic protocol. The
compounds tested were Phloroglucinol (P), Tannic
acid (TA) and Salicylic acid (SA), all used sepa-
rately at 30 or 300 lM. All compounds were
autoclaved as salts for 15 min (since their melting
points were all higher than 121 C) and were
added to autoclaved medium under sterile condi-
tions to the final concentrations indicated. Induc-
tion media included: no IBA (negative control),
10 mg l
1
IBA (positive control), and 10 mg l
1
IBA plus 30 and 300 lM of P or TA. Formation
media were devoid of IBA, P and TA, but sup-
plemented with activated charcoal. However, SA
was added at 30 and 300 lM at the formation
phase in the absence of activated charcoal. Positive
and negative controls devoid of activated charcoal
in the formation phase were also included to test
the effect of SA in the rooting response. SA con-
trols (SANHC and SAIBAC) were devoid of sal-
icylic acid.
The involvement of ethylene in the rooting re-
sponse was ex amined by adding silver nitrate, a
known ethylene action inhibitor (De Klerk et al.
1999), to the induction medium. Silver nitrate was
added at 25 and 50 lM both in the presence or ab-
sence of 10 mg l
1
IBA. Controls containing so-
dium nitrate in the same concentrations were
included to compensate for the effects of nitrate it-
self. The formation medium followed the composi-
tion of the original protocol.
Experiments were carri ed out in 15 ml glass flasks
containing 6 ml of culture medium, capped with a
double layer of aluminum foil. All experiments were
performed in a growth room with a photoperiod of
16 h of light (P.A.R. of 35 lmol m
2
s
1
at explant
level), provided by white fluorescent lamps, and
temperature of 27 ± 2 C, unless stated otherwise.
Flasks positions were changed daily to avoid minor
differences in irradiance. Two microcuttings were
used per flask, and treatments had 15 replicated
flasks, except for IAAasp, which had 11 replica-
tions. Experimental lay out was completely ran-
domized. Experiments were repeated independently
two to three times, yielding similar results.
Evaluated parameters and statistical analyses
Parameters evaluated were rooting percentage,
roots per rooted explant, mean rooting time, and
length of the longest root. Data was observed
every 2 days for 14 days after trans fer to forma-
tion medium, except for the length of the longest
root which was determined at the 14th day. All
white structure, with defined apex (polar), cylin-
3
drical and with at least 0.2 cm in length was con-
sidered an emitted root.
Statistical analyses (p £ 0.05) were done by
Analysis of Variance (ANOVA) followed, when-
ever appropriate, by Duncan test (software SSPS
for Windows, version 10.0.01). All data were
analyzed for normal distribution with the aid of
the same software to verify test suitability. Root-
ing percentage was transformed to log of 10 + 1
(Sokal and Rohlf 1981). Mean rooting time was
calculated as previous ly described (Fett-Neto et al.
2001); means with non-overlapping trust intervals
at p £ 0.05 were considered significantly differ-
ent.
Results and discussion
Auxin type
The best rooting percentage in E. saligna was
obtained with IBA and IAA, wher eas the control
devoid of auxin and the medium containing
IAAasp yielded the lowest responses. Cuttings
exposed to NAA had intermediate performance,
displaying significantly less rooting than their IBA
treated counterparts (Figure 1a). Eucalyptus glob-
ulus cuttings reached the highest rooting percent-
age after IBA treatment, even though IAA
exposure resulted in an intermediate performance
statistically equivalent to that of IBA (Figure 1a).
NAA and IAAasp treatment promoted rooting
only slightly. The comparison of rooting percent-
ages in control treatments of E. saligna and
E. globulus, 0 and 60%, respectively, confirms the
promptness to rooting of the former and the
recalcitrance of the latter (Figure 1a).
The better percent rooting respo nses of both
species when exposed to IBA could be related to
the relatively higher stability of this auxin, since
IAA is five times more readily photo-oxidized than
IBA (Nissen and Sutter 1990) and is more sus-
ceptible to enzymatic degradation (Ludwig-Muller
2000). However, the lower rooting percentages
reached by NAA (a very stable auxin) treated
cuttings compared to those cuttings exposed to
IAA do not support this possibility. The capacity
of IBA to be converted to IAA in vivo (Epstein and
Lavee 1984), functioning as a slow release reser-
voir of a more easily metabolized auxin, may be
the reason for the observed response. Studies with
Malus domestica cv. Jork 9 showed that rooting is
Figure 1. Rooting of microcuttings of Eucalyptus globulus (hatched bars) and E. saligna (empty bars) treated with different auxin
types. (a) Percent rooting; (b) Roots per rooted cutting; (c) Longest root length (mm); (d) Mean rooting time (days). Within each
species, bars sharing a letter are not different by a Duncan test (p £ 0.05). NH – no hormone control; IBA – indole butyric acid; IAA
– indole acetic acid; NAA – naphthaleneacetic acid; IAAasp – indole acetic acid aspartate conjugate.
4
more favored by IAA treatment than with NAA,
particularly in continuous exposure system (De
Klerk et al. 1997); it has been suggested that the
negative effect of NAA could be related to its
longer persistence compa red to other auxins,
remaining in tissues in free form and blocking root
emergence. Rooting experiments with microcut-
tings of Eucalyptus sideroxylon using IBA or NAA
in different concentrations (up to 10 lM) and
combinations under continuous exposure resulted
in more callus formation with IBA exposure
compared to NAA (Cheng et al. 1992). This was
not observed in the present experiments, probably
due to differences in auxin concentration, time of
exposure and species characteristics.
Both species of Eucalyptus had a higher number
of roots per rooted cutting when exposed to IBA
(Figure 1b). IAAasp treated cuttings and un-
treated ones of both species showed the lowest
response in terms of rooting percentage and root
density, (Figures 1a and b) suggesting that this
auxin conjugate is not hydrolyzed when exoge-
nously supplied or that its uptake is limited in
Eucalyptus.
The longest root length was similar in all
E. saligna treatments, except for IBA treated cut-
tings compared to the untreated controls (Fig-
ure 1c). This may be related to the shorter mean
rooting time in IBA treated c uttings allowing more
time for root elongation (Figure 1d). Although the
mean rooting time of IAA treated cuttings was
also sh ort, there was no significant effect on root
elongation, indicating that steady-state amounts of
this auxin may have been non-optimal for this step
of the rh izogenic process. In E. globulus, howeve r,
longest root length was significantly higher in IBA
treated cuttings compared to those exposed to
IAAasp an d NAA treatments (Figure 1c); IAA
treatment showed an inter mediate response. This
could be related to differences in the endogenous
concentrations of auxins between the species and/
or to a higher capacity of conversion of IBA in
IAA in E. globulus, so that reduction of auxin
concentration in the formation phase would favor
root growth and elongation (De Klerk et al. 1999).
The rooting response with IBA exposure in
E. globulus could also be influenced by its higher
capacity of being acropetally transported relative
to IAA (Ludwig-Muller 2000), which is the main
pathway of exogenous auxin influx to the plant,
along with transpiration-driven uptake via the
xylem. Auxin conjugation and/or metabolization
may be facilitated by this feature.
Differences in the response to auxins may reflect
variation in specific thres holds to trigger develop-
mental responses or to interfere in the metabolism
of other phytohormones, such as ethylene and
gibberellins (Ross and O’Neill 2001). Besides dif-
ferences in uptake and metabolism, auxin efficacy
may be affected by differential affinities for auxin
receptors involved in the rooting response.
Light intensity
Cuttings of E. saligna showed little response to the
various light intensities applied during the rooting
protocol. Only the treatment devoid of auxin un-
der 100 lmol m
2
s
1
had the rooting percentage
reduced (Figure 2a). Eucalyptus globulus cuttings
depended on the presence of exogenous auxin to
root, independently of the amount of irradiance
(Figure 2a); data on root density showed similar
response (Figure 2b). For E. saligna, auxin pro-
moted root density pe r rooted cutting in the
presence of light (Figure 2b). Overall these results
are in good agreement with previous data exam-
ining the effect of light (35 lmol m
2
s
1
) and
darkness in the two rooting phases of the same
species (Fett-Neto et al. 2001).
The inhibitory effect of higher irradiance on the
rooting percentage of E. saligna cuttings not ex-
posed to auxin could be partly related to photo-
induced changes in medium components
(Stanisopoulos and Hangarter 1990). Light in-
duced changes in cutting metabolism may also
have contributed to the rhizogenic response ob-
tained. Phenolic comp ound biosynthesis under
light can lead to inhibition of auxin basipetal
transport (Jacobs and Rubery 1988), changes in
peroxidase activity (Kevers et al. 1997) or auxin
protection in the wound zone (antioxidant activ-
ity) (De Klerk et al. 1999). A cytokinin-like com-
pound induced under high irradiance was
associated to rooting recalcitrance in Picea abies
cuttings (Bollmark and Eliasson 1990).
The positive effect of light on root density of
auxin-treated cuttings of E. saligna (Figure 2b) and
the trend towards higher rooting percentage in E.
globulus (Figure 2a) cuttings under the same con-
dition could be linked to greater auxin uptake from
medium due to the transpiration -driven mass flow
5
in light, as reported for other species (Jarvis and Ali
1984), or to light stimulation of young leaf devel-
opment and consequent increased availability of
endogenous auxin sources to supply the rooting
zone, as suggested for tomato cuttings (Tyburski
and Tretyn 2004). Higher light intensity and auxin
treatment favored root elongation in both species
examined in the present study, whereas in E. saligna
this response was also seen in cuttings without
auxin exposure (Figure 2c). Once again, these re-
sults agree with previously reported observations
on the rooting of the same species in darkness and
under one level of irradiance (Fett-N eto et al. 2001).
This response may reflect light-induced decrease in
auxin activity, allowing better root elongation.
The mean rooting time was reduced by light in
E. saligna cu ttings without significant effect of the
presence of auxin (Figure 2d). In E. globulus, this
parameter was not significantly affected in the
treatments showing rooting. Since in the present
investigation sucrose was exogenously supplied, it
is likely that light effects on adventitious root
development were related to phytohormone up-
take and metabolism, rather than carbon source
availability.
Apex removal
Apex removal did not affect the rhizogenic
response to exogenous IBA of any of the Euca-
lyptus species (data not shown). Lack of the main
endogenous source of auxin is therefore not lim-
iting to rooting when IBA is exogenously supplied
in appropriate concentration. Absence of the ter-
minal apex has not affected the rooting of auxin-
Figure 2. Rooting of microcuttings of Eucalyptus globulus (hatched bars) and E. saligna (empty bars) treated or not treated with IBA
grown under different irradiances. (a) Percent rooting; (b) Roots per rooted cutting; (c) Longest root length (mm); (d) Mean rooting
time (days). Within each species, bars sharing a letter are not different by a Duncan test (p £ 0.05). NH – no hormone control; IBA –
indole butyric acid treated; numbers at the end of treatment names refer to lmol m
2
s
1
of white light irradiance.
6
treated Pinus taeda and Picea abies cuttings eithe r
(Foster et al. 2000).
Phenolic adjuvants
The phenolic compounds tested had limited effects
on the rooting percentage and density of both
species, compared to the standard rooting proto-
col (Figure 3a and b). In E. globulus, the control
medium devoid of auxin in the induction phase
and activated charcoal in the formation phase did
not show rooting in the first experiment, suggest-
ing the presence of a rootin g inhibitor removable
by binding to activated charcoal. However, in a
duplicate experiment this treatment was equivalent
to the control devoid of auxin but containing
activated charcoal (data not shown). Another
noticeable aspect in this species was a trend toward
rooting percentage inhibition in the presence of
300 lM of salicylic acid during the root form ation
step (Figure 3a). Root elongation was inhibited in
E. saligna and a trend toward inhibition was seen
in E. globulus when cuttings were treated with
300 lM of salicylic acid (Figure 3c). Salicylic acid
is known to induce auxin catabolism by promoting
IAA oxidase activity (Kevers et al. 1997); it is
possible that, at the higher concentration of this
phenolic compound, auxin amounts were reduced
below the optimum for root formation and elon-
gation. Alternatively, uptake and transport of
salicylic acid may have reduced stomatal opening
(Raskin 1992) and affected transpiration-driven
auxin uptake.
In E. globulus, control treatments without auxin
yielded no rooting in the experiments depicted in
Figures 1 and 2, whereas a low but detectable
rooting percentage was observed in the experi-
ments shown in Figures 3 and 4. These differences
are within the expected experimental variation and
probably reflect slight differences in the rate of
rooting capacit y loss between seedling batches
used as cutting donors in the various experiments.
Tannic acid and phloroglucinol did not affect
significantly the rooting response of E. saligna and
E. globulus (Figur e 3a and b). Adventitious root-
ing of apple leaf disks was promoted by tannic acid
(between 0.1 and 100 lM) or phloroglucinol
(300 lM), presumably by increasing tissue com-
petence to respond to auxin or by protecting IAA
from oxidation (De Klerk et al. 1999). In a study
involving tw o apple rootstocks, M.9 (difficult-to-
root) and M .26 (easy-to-root), 1000 lMof
phloroglucinol combined with IBA significantly
promoted rooting of M.9 compared to auxin-alone
treatment, whereas M.26 was not affected by the
phenolic; the positive effect of phloroglucinol in
rooting was enhanced by previous exposure to the
compound (James and Thurbon 1981). The lack of
significant effects of phloroglucinol on Eucalyptus
adventitious rooting may reflect differences in
species-specific requirements for the process or the
need of longer exposure times prior to rooting it-
self in order to obtain a visible response.
Unlike rooting percentage and root density per
rooted cutting, E. globulus growth of the longest
root was significantly reduced by 300 lMof
phloroglucinol (Figure 3c), whereas no effect of
this compound was observed in E. saligna. This
suggests that root growth in E. globulus is more
sensitive to higher concentrations of remaining
auxin in the formation phase or that phloroglu-
cinol itself is inhibitory to root elongation in this
species. None of the species examined was signifi-
cantly affected in the mean rooting time by treat-
ment with phenolics compared to the controls
(data not shown).
Ethylene action inhibition
The overall effects of the ethylene action inhibitor
silver nitrate suggest that there is no significant
involvement of this phytohormone in the rhizo-
genic response of both Eucalyptus species. The
morphogenic responses observed are likely derived
directly from the action of auxin. Ethylene induced
by auxin exposure has not inhibited the rhizoge nic
response. Nitrat e itself had no effect on rootin g of
both species (Figure 4). However, in the present
experiments, the higher concentration of silver
nitrate showed a trend toward root number
reduction in E. saligna and a significant reduction
in the length of the longest root in E. globulus
(Figure 4b and c); these inhibitory effects could be
due to silver toxicity or to different sensibility of
the species in relation to the parameters number of
root differentiation sites and root elongation,
respectively. No effects of silver treatment were
observed in the mean rooting time. In contrast,
auxin induced adventitious rhizogenesis was
strongly promoted in apple microcuttings treated
7
with 10 lM of silver thiosulfate (an ethylene
action inhibitor) during the auxin exposure phase,
whereas the presence of aminocyclopropanic acid
(ACC, an ethylene precursor) inhibited rooting
(De Kler k et al. 1999).
In summary, the recalcitr ant rooting response of
E. globulus compared to E. saligna was evident in
all assays, as well as the central importance of
exogenous auxin for rooting promotion in the
former species. The results showed that the main
Figure 3. Rooting of microcuttings of Eucalyptus globulus (hatched bars) and E. saligna (empty bars) treated with IBA and different
phenolic compounds. (a) Percent rooting; (b) Roots per rooted cutting; (c) Longest root length (mm). Within each species, bars sharing
a letter are not different by a Duncan test (p £ 0.05). NH – no hormone control without phenolic addition; IBAC – indole butyric
acid control without phenolic addition; P – phloroglucinol; T – tannic acid; SA – salicylic acid; numbers in treatment names refer
to lM of phenolic compound. SANHC – salicylic acid control without SA, no IBA in the induction step and no activated charcoal in
the formation step; SAIBAC – salicylic acid control without SA, with IBA in the induction step and no activated charcoal in the
formation step. P and T were added during the induction phase, whereas SA was supplemented during the formation phase.
8
differences in stimulated rhizogenic response be-
tween the easy-to-root and difficult-to-root species
of Eucalyptus were related to auxin type. Auxins of
intermediate stability were more effective for
rooting than auxins of higher or lower stability,
particularly for the recalcitrant species. In both
species, more notably in the easy-to-root, exoge-
nous auxin-induced rhizogenesis was more effec-
tive in the presence of higher light intensities,
possibly due to increased auxin influx. In the
presence of exogenous auxin, the absence of the
meristematic apex or externally supplied phenolics
did not significantly affect the rhizogenic capacity
of any of the species. Finally, the morphogenic
response of both species is apparently the direct
result of auxin action, with ethylene playing a
minor role.
Taken together, the results indicate that there
are fundamental differences between the adventi-
tious rooting in Malus and Eucalyptus, which
should be considered in attempts to model this
developmental process in woody species cultivated
in vitro.
Acknowledgments
We thank Teotoˆ nio F. de Assis (Aracruz S.A.) for
the gift of seeds. Financial support was from
Brazilian agencies: grants to AGFN (CNPq, FA-
PERGS) and scholarships to CMF (CAPES).
References
Bollmark M. and Eliasson L. 1990. A rooting inhibitor present
in Norway spruce seedlings grown at high irradiance – a
putative cytokinin. Physiol. Plant. 80: 527–533.
Brock T.G. and Kauffman P.B. 1991. Growth regulators: An
account of hormones and growth regulation. In: Steward
F.C. (ed.), Plant Physiology – A Treatise, Vol X. Academic
Press, San Diego, pp. 277–340.
Figure 4. Rooting of microcuttings of Eucalyptus globulus
(hatched bars) and E. saligna (empty bars) treated with an
ethylene action inhibitor in the induction step. (a) Percent
rooting; (b) Roots per rooted cutting; (c) Longest root length
(mm). Within each species, bars sharing a letter are not different
by a Duncan test (p £ 0.05). NH – no hormone control; IBA –
indole butyric acid treated; Na – sodium nitrate added; Ag –
silver nitrate added; numbers in treatment names refer to lMof
nitrate salt.
b
9
Brown D.E., Rashotte A.M., Murphy A., Normanly J., Tague
B.W. and Muday G.K. 2001. Flavonoids act as negative
regulators of auxin transport in vivo in Arabidopsis. Plant
Physiol. 126: 524–535.
Cheng B., Peterson C.M. and Mitchell R.J. 1992. The role of
sucrose, auxin and explant source on in vitro rooting of
seedling explants of Eucalyptus sideroxylon. Plant Sci. 87:
207–214.
Crozier A., Kamiya Y, Bishop G. and Yokota T. 2000. Bio-
synthesis of hormones and elicitor molecules. In: Buchanan
B.B., Gruissem W. and Jones R.L. (eds), Biochemistry and
Molecular Biology of Plants. American Society of Plant
Physiologists, Rockville, pp. 850–929.
Curir P., Vansumere C.F., Termini A., Barthe P., Marchesini
A. and Dolci M. 1990. Flavonoid accumulation is correlated
with adventitious roots formation in Eucalyptus gunni Hook
micropropagated through axillary bud stimulation. Plant
Physiol. 92: 1148–1153.
De Klerk G.-J., Krieken W.V.D. and Jong J. 1999. The for-
mation of adventitious roots: new concepts, new possibilities.
In Vitro Cell Dev. Biol. 35: 189–199.
De Klerk G.-J., Brugge J.T. and Marinova S. 1997. Effective-
ness of indoleacetic acid, indolebutyric acid and naphtha-
leneacetic acid during adventitious root formation in vitro in
Malus Jork 9. Plant Cell Tiss. Org. Cult. 49: 39–44.
Dharmasiri N. and Estelle M. 2004. Auxin signaling and reg-
ulated protein degradation. Trends Plant Sci. 9: 302–308.
Epstein E. and Lavee S. 1984. Conversion of indole-3-butyric
acid to indole-3 acetic acid by cuttings of grapevine (Vitis
vinifera) and olive (Olea europea). Plant Cell Physiol. 25: 697–
703.
Faivre-Rampant O., Kevers C., Bellini C. and Gaspar T. 1998.
Peroxidase activity, ethylene production, lignification and
growth limitation in shoots of a rooting mutant of tobacco.
Plant Physiol. Biochem. 36: 873–877.
Fett-Neto A.G., Fett J.P., Goulart L.W.V., Pasquali G., Ter-
mignoni R.R. and Ferreira A.G. 2001. Distinct effects of auxin
and light on adventitious root development in Eucalyptus
saligna and Eucalyptus globulus. Tree Physiol. 21: 457–464.
Fett-Neto A.G., Teixeira S.L., Da Silva E.A.M. and Sant’Anna
R. 1992. Biochemical and morphological changes during in
vitro rhizogenesis in cuttings of Sequoia sempervirens(D
Don). Engl. J. Plant Physiol. 140: 720–728.
Foster G.S., Stelzer H.E. and McRal J.B. 2000. Loblolly pine
cutting morphological traits: effects on rooting and field
performance. New For. 19: 291–306.
Gaspar T. and Thorpe T.A. 1977. Changes in isoperoxidases
during differentiation in cultured tobacco epidermal layers.
Acta Hortic. 78: 61–73.
Gonza
´
lez A., Tame
´
s R.S. and Rodriguez R. 1991. Ethylene in
relation to protein, peroxidase and polyphenol oxidase
activities during rooting in hazelnut cotyledons. Physiol.
Plant. 83: 611–620.
Jacobs M. and Rubery P.H. 1988. Naturally occurring auxin
transport regulators. Science 241: 346–349.
James D.J. and Thurbon I.J. 1981. Phenolic compounds and
other factors controlling rhizogenesis in vitro in the apple
rootstocks M.9 and M.26. Z. Pflanzenphysiol. 105: 1–10.
Jarvis B.C. and Ali A.H.N. 1984. Irradiance and adventitious
root formation in stem cuttings of Phaseolus aureus Roxb.
New Phytol. 97: 31–36.
Kevers C., Hausman J.F., Faivre-Rampant O., Evers D. and
Gaspar T. 1997. Hormonal control of adventitious rooting:
progress and questions. Angew. Bot. 71: 71–79.
Lee T.T., Starrat A.N. and Jevnikar J.J. 1982. Regulation of
enzymic oxidation of indole-3-acetic acid by phenols: struc-
ture-activity relationships. Phytochemistry 21: 517–523.
Ludwig-Mu
¨
ller J.L. 2000. Indole-3-butyric acid in plant growth
and development. Plant Grow. Regul. 32: 219–230.
Moncousin C., Favre J.M. and Gaspar T. 1988. Changes in
peroxidase activity and endogenous IAA leves during
adventitious rooting in vine cuttings. In: Kuta
´
cek M., Ban-
durski R.S. and Krekule J. (eds.), Physiology and Biochem-
istry of Auxins in Plants. Academic Publishing, Prague, pp.
331–337.
Muday G.K. and DeLong A. 2001. Polar auxin transport:
controlling where and how much. Trends Plant Sci. 6: 535–
542.
Nissen S.J. and Sutter E.G. 1990. Stability of IAA and IBA in
nutrient medium of several tissue culture procedures. Hort-
Science 25: 800–802.
Raskin I. 1992. Role of salicylic acid in plants. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 43: 439–463.
Reid D., Beall F.D. and Pharis R.P. 1991. Environmental cues
in plant growth and development. In: Steward F.C. (ed.),
Plant Physiology – A Treatise, Vol X. Academic Press, San
Diego, pp. 65–181.
Ross J. and O’Neill D. 2001. New interactions between classical
plant hormones. Trends Plant Sci. 6: 2–4.
Sokal R.R. and Rohlf F.J. 1981. Biometry. W.H. Freeman, San
Francisco 859pp.
Stasinopoulos T.C. and Hangarter R.P. 1990. Preventing pho-
tochemistry in culture media by long-pass light filters growth
of cultured tissues. Plant Physiol. 93: 1365–1369.
Tyburski J. and Tretyn A. 2004. The role of light and polar
auxin transport in root regeneration from hypocotyls of to-
mato seedling cuttings. Plant Growth Regul. 42: 39–48.
Visser E.J.W., Cohen J.D., Barendse G.W.M., Blom
C.W.P.M. and Voesenek L.A.C.J. 1996. An ethylene-medi-
ated increase in sensitivity to auxin induces adventitious root
formation in flooded Rumex palustris Sm. Plant Physiol.
112: 1687–1692.
10