Tree Physiology 23, 1247–1254
© 2003 Heron Publishing—Victoria, Canada
Developmental stages during the rooting of in-vitro-cultured Quercus
robur shoots from material of juvenile and mature origin
N. VIDAL,1 G. ARELLANO,1,2 M.C. SAN-JOSÉ,1 A.M. VIEITEZ1 and A. BALLESTER1,3
1
Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain
2
Present address: Especialidad de Fruticultura-IREGEP-Colegio de Postgraduados, Km. 36.5 carretera México-Texcoco, Montecillo Edo. de México,
CP 56230, México
3
Author to whom correspondence should be addressed ([email protected])
Received March 19, 2003; accepted May 26, 2003; published online November 17, 2003
Summary In-vitro-cultured shoots of clones initiated from
shoots of the basal parts (BS) and the crown (C) of two mature
Quercus robur L. trees were subjected to rooting experiments
to relate rooting with shoot topophysical origin. The BS shoots
exhibited morphologically juvenile characteristics and rooted
more easily after indole-3-butyric acid (IBA) treatment than C
shoots. When naphthylphthalamic acid (NPA) was applied to
BS shoots, rooting capacity decreased and root emergence was
delayed at least 2 days compared with shoots treated with IBA
only. During the first days of the rooting process, endogenous
indole-3-acetic acid (IAA) concentration was higher in C
shoots than in BS shoots, regardless of whether the shoots were
treated with NPA. Mitotic figures were observed in cells from
the basal part of both BS and C shoots 24 h after IBA treatment.
After 4 days of IBA treatment, the occurrence of histological
events differed between BS shoots and C shoots. Cells of BS
shoots became meristematic, giving rise to meristemoids and
root primordia, whereas no differentiation of root meristemoids occurred in cells of C shoots. Thus, although adult oak
material (C shoots) is capable of responding to the initial stimulus of auxin during the adventitious rooting process, the endogenous IAA concentration is not the factor limiting rooting
in adult material.
Keywords: anatomical study, endogenous auxin content, oak,
root primordium formation.
Introduction
Maturation in plant species is an ontogenetic process in which
progressive changes in particular attributes take place. Although flowering capacity is the factor indicating that the maturation stage has been achieved, other attributes (e.g., the
shape of the leaves, shoot orientation, stem pigmentation, and
rooting capacity) are also modified through the phase change
(Hackett 1985). Rooting capacity is one of the economically
important factors involved in phase change. In forest tree species, lack of rooting hinders the clonal propagation of material
derived from genetic improvement programs.
Hackett and Murray (1996) concluded that events associ-
ated with phase change may be studied from either an epigenetic perspective (comparison of tissues of the same genotype
but at different ontogenetic states) or a genetic perspective (in
this case, a mutant genotype is required). Ballester et al. (1999)
used an epigenetic approach to study the rooting process associated with phase change of chestnut shoots cultured in vitro.
According to De Klerk et al. (1999), adventitious root formation in cuttings and shoots occurs in four steps: cell dedifferentiation, induction, root primordia development and root
emergence. At each step, auxin (endogenous or exogenously
applied) plays a central role. A high endogenous auxin concentration at the beginning of the rooting process is normally
associated with a high rooting rate (Jay-Allemand et al. 1995,
Blazkova et al. 1997, Caboni et al. 1997), although a negative
relationship has also been reported (Feito et al. 1996). When
exogenous auxin is applied to induce rooting, the endogenous
auxin concentration usually reaches a peak some hours or days
after wounding (Gaspar et al. 1996, Gatineau et al. 1997), coinciding with the initiation of the rooting process. However, in
some cases, the peak in auxin concentration has not been observed, or a continuous increase has been detected (Berthon et
al. 1989, Label et al. 1989). These contradictory results can be
accounted for by the different experimental systems used.
Most models do not compare rooting capacity in tissues at different ontogenetic stages of development (cf. Hackett and
Murray 1996). In addition, a lack of knowledge of auxin catabolism, conjugation and transport, and the identification of appropriate receptors in specific cells, compound the difficulties
associated with the experimental design of in vitro rooting
studies.
Based on a model used to study the in vitro rooting process
in chestnut (Ballester et al. 1999), we developed an in vitro
experimental system for oak (Quercus robur L.) designed to
correlate different aspects of the rooting process with the ontogenetic state of the plant material. In-vitro-derived oak shoot
cultures were established from material simultaneously collected from the crown (C) and basal (BS) shoots of 100- to
300-year-old trees (Vieitez et al. 1994). Based on current
knowledge of phase change in woody species (Hackett 1985),
we predicted that BS shoots would exhibit a juvenile pheno-
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VIDAL, ARELLANO, SAN-JOSÉ, VIEITEZ AND BALLESTER
type (high rooting capacity), whereas C shoots would respond
like mature material (low rooting capacity). Specifically, we
studied: (1) morphological differences between in-vitro-cultured shoots derived from BS and C shoots; (2) rooting characteristics of BS and C shoots, including the effect of naphthylphthalamic acid (NPA, an inhibitor of auxin transport), the
kinetics of the rooting process and the anatomical events leading to formation of root primordia; and (3) changes in endogenous auxin concentration during the first days of the rooting
process.
phase). Ten ml of water was added to the controls. The shoots
were in contact with the NPA solution for 0, 1, 2, 3 or 5 days,
but after 24 h, the shoots were transferred to auxin-free medium and the NPA was replaced by fresh NPA for 2, 3 or
5 days. In this experiment, we used 100-ml glass jars each containing 30 ml of rooting medium.
In all rooting experiments, each jar contained six shoots and
at least three jars were used per experiment. Rooting rate (%),
the number of roots per rooted shoot and the time elapsed before the appearance of the first root per shoot were recorded
28 days after the beginning of the experiment. All experiments
were repeated at least three times.
Materials and methods
Anatomical study
Plant material
Shoot culture lines BS and C were established in vitro from
shoots collected from the base and crown, respectively, of a
300-year-old Quercus robur L. tree, clone Sainza (Vieitez et
al. 1994). In vitro BS and C shoot cultures of clone NL100,
established from a 100-year-old tree by the same procedure,
were supplied by Dr. Peter Evers (IBN-DLO, Wageningen,
The Netherlands) through COST 822 (European Commission)
in 1994. The BS and C lines have been routinely subcultured in
GD medium (Gresshoff and Doy 1972) supplemented with
30 g l –1 sucrose, 6.5 g l –1 agar (Vitroagar, Hispanlab, Spain)
and 0.44 µM N 6-benzyladenine (BA). Shoot explants, with the
apical 2 mm removed, were placed horizontally in 500-ml
glass jars containing 70 ml of multiplication medium and were
transferred to fresh medium every 2 weeks during the 4-week
multiplication cycle (Vieitez et al. 1993, Sánchez et al. 1996).
Morphological study
Microshoots derived from BS and C shoots of Clone Saínza,
isolated at the end of the subculture period, were subjected to
morphological analyses by means of computerized digital image analysis. Length, stem base diameter, number of leaves
and number of internodes were recorded per shoot. Area, total perimeter, convex perimeter, length, width, roundness
(4π(area)/(perimeter) 2 ), aspect ratio (width/length) and perimeter ratio (convex perimeter/total perimeter) were recorded
per leaf. Ten shoots (stem and leaves) per line were sampled,
and the experiment was repeated three times.
Rooting
Microshoots (20 mm in length) derived from BS and C shoots
of the Saínza and NL100 clones were harvested at the end of
the multiplication step. To induce rooting, the shoots were
placed on GD medium with 1/3-strength macronutrients, and
supplemented with 5.5 g l –1 agar and either 0 (control) or
0.12 mM IBA. After 24 h, shoots were transferred to auxinfree medium of the same composition supplemented with 6.5 g
l –1 agar. In this experiment, 300-ml glass jars, each containing
60 ml of rooting medium, were used.
To study the effect of NPA on rooting, microshoots derived
from BS shoots of Clone Saínza were induced to root on
0.12 mM IBA medium supplemented with 10 ml of an aqueous filter-sterilized solution of 200 µM NPA (added as double
Basal segments (0.5 cm) from lines BS and C of Clone Saínza
were collected from shoots induced to root with IBA. Samples
were collected daily from Day 0 (controls) to Day 10 and then
at Days 12, 14, 18, 20 and 28 after the start of the experiment.
Samples were fixed in formalin:glacial acetic acid:50% ethanol (5:5:90, v/v), dehydrated through a graded n-butanol series
and embedded in paraffin. Transverse sections (10 µm) were
cut with a rotatory microtome and stained with safranin-fast
green (Jensen 1962).
Auxin determination
Samples of lines BS and C of Clones Saínza and NL100 were
collected 0, 0.5, 1, 2, 3 and 4 days after the rooting treatment
(with or without IBA). Additional samples of lines BS and C
of Clone Saínza were collected from the NPA experiment up to
Day 8. For all samples, leaves were discarded and shoots were
divided into two parts: the lowermost 1 cm of the stem base
(hereafter basal section) and the rest of the shoot (hereafter
apical section). In the NPA experiment, only basal sections
were analyzed. Both sections were immersed in liquid nitrogen and lyophilized. Extraction and determination of auxins
(IAA, IAAsp (indole-3-acetylaspartic acid) and IBA) was performed according to Nordström and Eliasson (1991) with the
modifications reported by Ballester et al. (1999). Briefly, lyophilized plant material (100 mg) was extracted in 5 mM
potassium phosphate buffer (pH 6.5), purified through
Chromabond C18 columns (100 mg) and filtered before
HPLC analysis. The methanolic extracts were injected into a
Waters HPLC system (column: Hypersil ODS, 6 cm long; mobile phase: 84% A (acetonitrile:acetic acid:water (10:2:88)),
16% B (acetonitrile) for IAA and IBA, and 95% A, 5% B for
IAAsp; flow: 1 ml min –1). A Waters fluorescence detector was
set at excitation and emission wavelengths of 292 and 360 nm,
respectively). Data are means of three replicates of plant material, and the whole experiment was repeated at least twice.
Data were subjected to analysis of variance (ANOVA). The
identity of the IAA peak was confirmed by gas chromatography–mass spectrometry analysis performed according to Alvarez et al. (1989) with the slight modifications reported by
Ballester et al. (1999).
Statistical analysis
Data were subjected to one-way ANOVA. In some cases, the
TREE PHYSIOLOGY VOLUME 23, 2003
ROOTING OF OAK SHOOTS
least significant difference test at the 5% probability level was
used to compare means. Percentage data were subjected to
arcsine square-root transformation before ANOVA.
1249
Table 2. Rooting of in-vitro-cultured shoots derived from basal (BS)
and crown (C) shoots of Quercus robur Clones NL100 and Saínza on
medium supplemented with 0.12 mM IBA for 24 h. Data were recorded on Day 28 of the experiment. Within a clone, means followed
by the same letter are not significantly different (P = 0.001).
Results
The morphological characteristics of in-vitro-cultured shoots
derived from BS and C shoots of the Saínza clone are shown in
Table 1. The C shoots were shorter than the BS shoots, but the
base diameter and number of nodes of C shoots were higher. In
contrast, the shoot and internodal sections were larger in BS
shoots than in C shoots. The majority of parameters evaluated
for the leaves were significantly higher in BS shoots than in C
shoots. There was no difference in the roundness and aspect
ratio, but the perimeter ratio was higher in C shoots than in BS
shoots. The roundness and aspect ratio data suggest that there
were only subtle changes in the shape of the leaf with maturity.
The BS shoots had greater rooting capacity than the C
shoots (Table 2), confirming the greater degree of juvenility of
the BS material. Clear clonal differences were also observed.
The C shoots from Clone Saínza rooted poorly (2.2%),
whereas rooting of C shoots from Clone NL100 was 64.7%.
Based on the large difference in rooting capacity of BS and C
shoots of Clone Saínza, we selected shoots of this clone for
further study.
The presence of NPA affected rooting capacity and delayed
the onset of rooting in BS shoots of Clone Saínza (Table 3).
Rooting capacity declined as the NPA application period increased, reaching 50% inhibition relative to the control after
5 days of NPA treatment (Figure 1). We observed a 1–3 day
delay in the onset of the rooting process in response to NPA
compared with the controls (Figure 1). Control shoots reached
maximum rooting capacity 20 days after the start of the experiment, and this capacity remained constant until the end of the
trial. In response to a 3-day treatment with NPA, maximum
Clone
Line
Rooting (%)
Number of roots
NL100
BS
C
100 ± 0.0 a
64.7 ± 9.9 b
5.0 ± 0.7 a
1.8 ± 0.3 b
Saínza
BS
C
73.4 ± 11.1 a
2.2 ± 3.0 b
2.7 ± 0.5 a
0.4 ± 0.5 b
Table 3. Effect of number of days of naphthylphthalamic acid (NPA)
application on the rooting of in-vitro-cultured shoots derived from
basal shoots of Quercus robur Clone Saínza. The shoots were subjected to 0.12 mM IBA treatment for 24 h. Data were recorded on
Day 28 of the experiment. Within a column, means followed by the
same letter are not significantly different (P = 0.05).
Days of NPA treatment
Rooting (%)
Number of roots
0 (Control)
1
2
3
5
87.3 ± 4.4 d
71.4 ± 9.5 cd
63.2 ± 7.5 bc
47.5 ± 5.1 ab
41.0 ± 6.5 a
2.6 ± 0.2 bc
2.4 ± 0.3 bc
1.9 ± 0.2 ab
1.7 ± 0.3 ab
1.2 ± 0.08 a
rooting capacity was reached 8 days after the start of the experiment. A 5-day treatment with NPA markedly reduced the
rooting process compared with the control and the 3-day NPA
treatment.
Histological analysis showed that, on Day 0, before IBA application, BS and C shoots had similar anatomical structures,
Table 1. Morphological characteristics of in-vitro-cultured shoots derived from basal (BS) and crown (C) shoots of Quercus robur Clone
Saínza. Data were recorded at the end of the multiplication culture.
Characteristic
BS
C
Stem
Length (mm)
Base diameter (mm)
Number of leaves
Number of nodes
24.3 ± 1.3
1.3 ± 0.1
9.5 ± 0.9
9.3 ± 0.7
20.8 ± 0.8
1.8 ± 0.1
9.7 ± 0.3
11.1 ± 0.4
*1
*
ns1
*
Leaves
Area (mm2)
Perimeter (mm)
Convex perimeter (mm)
Length (mm)
Width (mm)
Roundness
Aspect ratio
Perimeter ratio
51.4 ± 6.9
38.5 ± 3.1
28.1 ± 2.0
13.3 ± 0.8
5.4 ± 0.4
0.45 ± 0.01
0.40 ± 0.07
0.77 ± 0.01
29.6 ± 2.0
26.9 ± 1.3
22.1 ± 1.0
10.4 ± 0.5
4.0 ± 0.1
0.51 ± 0.01
0.38 ± 0.01
0.83 ± 0.008
*
*
*
*
*
ns
ns
*
1
An asterisk denotes significant at P = 0.05; and ns = not significant.
Figure 1. Effect of 200 µM naphthylphthalamic acid (NPA) on rooting
of in-vitro-cultured oak shoots derived from a basal (BS) shoot line of
Clone Saínza. The shoots were treated with the NPA solution (double
phase) for 3 or 5 days.
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VIDAL, ARELLANO, SAN-JOSÉ, VIEITEZ AND BALLESTER
including a cambial zone with two to three layers of flattened
cambial cells between the xylem and phloem tissues of collateral vascular bundles. As seen in transections, the vascular system was surrounded by a discontinuous ring of sclerenchyma
formed by groups of phloem fibres (Figure 2A).
After 24–48 h of auxin treatment, certain cells in the
phloem region, interfascicular parenchyma and inner cortex
became dedifferentiated, exhibiting prominent central nuclei
with a large nucleolus. Mitotic figures, with mostly periclinal
division planes, were observed in these cell types after 24 h of
auxin treatment (Figure 2B), increasing the number of mitotic
and dedifferentiating cells in the following 2–3 days in both
BS and C shoots (Figures 2C and 2D). From Day 4 onward, the
occurrence of histological events differed between the BS and
C microcuttings.
In BS microcuttings, during Days 4–5, certain cells that remained less differentiated divided and formed small groups of
cells exhibiting meristematic characteristics: isodiametric,
densely stained cells with a high nucleolus-to-cell area ratio.
These localized meristematic clusters developed by both periclinal and anticlinal divisions and are assumed to make up the
organization of meristemoids (Torrey 1966). Meristemoids
appeared to originate from cambial derivatives, especially ray
parenchyma cells, and they generally developed in the outermost region of phloematic tissues close to the sclerenchymatic
ring (Figure 2E). The involvement of dedifferentiating cells of
Figure 2. Transverse sections
of the stem base of oak microcuttings derived from basal
(BS) and crown (C) shoots of
Clone Saínza 0–14 days after
indole-3-butyric acid (IBA)
root inductive treatment. (A)
Anatomical structure of the
stem of a BS shoot at the time
of excision (Day 0). (B) Stem
base of a C shoot 24 h after
treatment, showing early cell
activation and a periclinal division (arrow) in the cambial
zone. (C–D) Three days after
inductive treatment of a BS
shoot (C) and a C shoot (D),
showing an increasing number
of cambial derivative cells involved in dedifferentiation and
division (arrows). (E) Five
days after treatment: organization of meristemoids in the
outermost region of the
phloem tissues of a BS microcutting. (F) Day 8: early root
primordia (dome-like structures) pushing into the cortex
through the sclerenchymatic
fibre ring of a BS microcutting. (G) Day 11: protruding root primordia and
emerging adventitious root
through disaggregated callus
tissue of a BS shoot. Note the
differentiation of the root vascular system. (H) Day 14 after
inductive treatment, showing
callus formation mainly derived from the cortex of a C
microcutting. Abbreviations:
c = callus; m = meristemoid;
ph = phloem; s = sclerenchyma ring; and x = xylem.
Scale bars: 150 µm in A;
24 µm in B; 32 µm in C and D;
95 µm in E and F; 380 µm in
G; and 476 µm in H.
TREE PHYSIOLOGY VOLUME 23, 2003
ROOTING OF OAK SHOOTS
1251
Figure 3. Time course of
changes in concentrations of
indole-3-acetic acid (IAA) and
indole-3-acetylaspartic acid
(IAAsp) in basal and apical
sections from basal (BS) and
crown (C) shoots of Clones
Saínza and NL100 during the
first days after root induction
treatment. Bars represent ±
SE.
the surrounding inner cortex parenchyma contributing to the
buildup of the meristemoid was also evident, and some meristemoids originated entirely from inner cortex cells.
On Days 6–8, observation of BS samples revealed that root
meristemoids had differentiated into root primordia by a po-
larization of cell division in the distal end cells of the meristemoids, resulting in typically dome-shaped root primordia
(Figure 2F). This was coincident with the outer region of the
meristemoid tissue forcing the cortex through the sclerenchymatic fiber ring. Root primordia started to differentiate their
Figure 4. Effects of
naphthylphthalamic acid
(NPA) on endogenous concentrations of indole-3-acetic acid
(IAA) and indole-3-acetylaspartic acid (IAAsp) in the
basal section of in-vitro-cultured shoots derived from
basal (BS) and crown (C)
shoots of Clone Saínza after
root induction treatment. A
200 µM NPA solution was
added for 5 days. Bars represent ± SE.
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VIDAL, ARELLANO, SAN-JOSÉ, VIEITEZ AND BALLESTER
own vascular tissues as well as the root cap, whereas their distal ends grew through the cortex. The first adventitious roots
emerged at the surface 11 days after auxin treatment, and they
had developed a vascular system (Figure 2G) that was continuous with that of the stem by the time they emerged. In addition,
there was significant cell proliferation in the basal zone of BS
microcuttings, affecting the tissues directly related to the development of root primordia, i.e., the phloem and the cortex. In
the latter tissue, cell proliferation gave rise to disaggregated
callus tissue through which most adventitious roots protruded
(Figure 2G) within 16 days after the start of the rooting experiment.
From Day 4 of IBA treatment of C microcuttings, the number of mitotic figures in the phloem and inner cortex appeared
to be lower than in the corresponding material of BS origin (although no quantitative data were recorded), and no differentiation of root meristemoids occurred. By Days 4–8, cell division
was confined to the cambial zone, with the cutting-off of radial
files of a few cambial derivatives on the phloem side and xylem side, resulting in the evident differentiation of new lignified xylem elements. From Day 10 onward, no mitotic figures
were observed in the cambium–phloem region, which exhibited cells with condensed nuclei, whereas the accumulation of
phenolic substances in vascular ray cells was also evident. By
this time, although many of the C samples started to dry out, a
number of microcuttings developed a visible callus in their
basal zone by proliferation of the cortex cells (Figure 2H).
Changes in endogenous concentrations of IAA and IAAsp
over the first 4 days of rooting (when histological changes
leading to meristemoid formation occurred) are shown in Figure 3. During the in vitro rooting process in Clone Saínza, the
endogenous IAA concentration was always higher in C microshoots (difficult-to-root shoots) than in BS microshoots, regardless of whether the analysis was carried out on the basal or
apical part of the shoot. In Clone NL100, however, in which C
microshoots showed high rhizogenic capacity (64.7%), the endogenous IAA concentration was similar in both BS and C
microshoots, in both basal and apical parts of shoots (Figures 3A and 3B). In both clones, the endogenous IAA concentration was always much higher in bases of shoots than in
apices. In all cases, a notable increase in IAA concentration
was observed 24 or 48 h after the start of the rooting process,
which then remained constant or subsequently declined. When
analyses were carried out on control shoots (without IBA treatment in root induction), the quantity of detected IAA was low
(< 2.0 nmol g –1) and remained more or less constant throughout the 4 days (Vidal 2002). Contrary to the changes observed
for IAA, the IAAsp concentration was higher in BS microshoots that in C microshoots in the basal part of the shoot, and
was the same or slightly higher in the apical part of the shoot.
In both the apical and basal parts of the shoot, the IAAsp concentration was lower than the IAA concentration (Figures 3C
and 3D). Compared with IAA, the time course of changes in
IAAsp concentration was similar but less pronounced, with a
peak 1, 2 or 3 days after the onset of the rooting process.
In BS microshoots of both clones, IBA concentration
showed a pronounced peak 12–24 h after the start of the root-
ing process and then declined to a negligible concentration in
the ensuing days. The IBA concentration, when detectable,
was always lower in C microshoots than in BS microshoots
(Vidal 2002).
The effects of a 5-day treatment with NPA on IAA and
IAAsp concentrations are shown in Figure 4. The time courses
of changes in IAA and IAAsp concentrations in the presence
of NPA were similar to those observed when rooting took
place in the presence of IBA alone. At 24 h after the onset of
rooting, there was a peak in IAA (Figures 4A and 4B) or
IAAsp concentration (Figures 4C and 4D) with a subsequent
more or less pronounced decline on all days sampled thereafter. In BS microshoots treated with NPA, the endogenous IAA
concentration was higher than in the controls, which parallels
the difference in endogenous IAA concentration between BS
and C microshoots (Figure 3A). That is, the NPA-treated BS
microshoots exhibited an increase in endogenous IAA concentration that resembled the response of C shoots. In control
shoots, the peak in IAA concentration also occurred 24 h after
the onset of rooting. When NPA was applied to C shoots of low
rooting capacity, IAA concentration increased and the appearance of the observed peak was delayed 12 h with respect to the
control. These results indicate that NPA promotes an increase
in endogenous IAA concentration (possibly as a consequence
of inhibition of IAA transport) that is associated with a reduction in rooting capacity of the NPA-treated microshoots. The
IAAsp concentration in BS shoots was unaffected by NPA.
Discussion
In our oak clones, morphological characteristics of the shoots
and their proliferation and rooting capacity can be used as
markers for differentiating between explants of juvenile and
adult origins. Under our experimental conditions and after
9 years of subculture, shoots taken from the base and crown of
the same tree maintained their own topophysical characteristics, providing an efficient system for studying phase change
in woody species. Some authors (Webster and Jones 1989) associate an increase in number of subcultures with a rejuvenation process in the explants, whereas others (De Klerk 2002)
consider that the reverse transition, from the juvenile to the
adult state, can also occur with an increase in number of subcultures. We conclude that these relationships should not be
generalized, because neither relationship was observed in either the present study of oak or in chestnut (Ballester et al.
1999). Based on variability in rooting rates among different
assays, McGowran et al. (1998) concluded that the rooting capacity of Q. robur and Q. petraea (Matt.) Liebl. shoots was unsuitable as a marker for differentiating between the juvenile
and adult material. However, we found that rooting can serve
as a suitable marker of phase change, provided that the experimental system used ensures that variability among assays affects juvenile and adult material equally.
An experimental system similar to the model we have developed for oak has been described for Pinus taeda L. (Diaz-Sala
et al. 1996), in which cuttings from hypocotyls taken from
seedlings 50 days after germination root readily, whereas
TREE PHYSIOLOGY VOLUME 23, 2003
ROOTING OF OAK SHOOTS
epicotyl cuttings from the same seedlings root infrequently.
Nevertheless, our experimental model has the advantage that
the root formation zone corresponds to the same tissue regardless of the plant material used, which is not the case with the
loblolly pine system. We observed that oak explants derived
from basal shoots and crown shoots (BS and C, respectively)
had similar anatomical characteristics. At the start of the rooting process, cellular divisions were observed in both BS and C
explants in certain cells from the phloem, interfascicular parenchyma and internal cortex. Thus, there appears to be no
specific type of cell in either the BS or C explant that would
make it possible to distinguish which explant is going to be
able to form root primordia. Four days after the start of rooting, dividing cells from BS microshoots became meristematic
and grouped together to form meristemoids; however, in cells
from C microshoots, cell division was confined to the cambial
zone, resulting in the formation of new xylem elements, and
subsequently callus instead of meristemoids. Our results refute the hypothesis that adult material does not respond to the
initial stimulus of auxin, as has also been demonstrated in
Pinus strobus L. (Goldfarb et al. 1998), chestnut (Ballester et
al. 1999) and Pinus taeda (Greenwood et al. 2001). We conclude that loss of rooting ability in mature oak shoots in response to auxin is not due to a lack of initial auxin response.
It is generally accepted that a higher concentration of endogenous auxin in the base of the cutting results in higher rooting capacity (Blazkova et al. 1997). This relationship does not
hold for oak, or for other species such as apple (James 1983),
chestnut (Ballester et al. 1999) or the rac mutant of tobacco
(Faivre-Rampant et al. 2001). In all of these studies, there was
a higher concentration of endogenous auxin in shoots that
showed low rooting capacity after IBA-induced rooting. When
IBA was not applied to oak microshoots during the rooting
process, endogenous IAA concentrations remained low
throughout the in vitro rooting process. Although NPA application did not nullify root initiation by IBA, it caused a reduction in rooting capacity and, more importantly, a delay in root
formation. Furthermore, an increase in endogenous IAA concentration was observed in NPA-treated shoots. Thus, auxinmediated rooting in oak shoots seems to comprise several
phases: (1) IBA treatment is necessary to induce rooting; (2)
the effect of IBA treatment is not fully countered by the application of NPA; (3) the start of cell division in response to endogenous IAA, regardless of the ontogenetic state of the
material, is the first visible anatomical change; (4) only cells
from juvenile tissues continue to respond to the auxin stimulus—they become meristematic and have the capacity to form
root primordia; and (5) mature-state-tissue cells form callus
instead of root primordia in the presence of IAA.
Although endogenous IAA concentration does not seem to
be the limiting factor in the rooting of adult oak material, it is
possible that a lack of appropriate receptors prevents IAA
from acting on the cells of this material. Currently, the best
characterized auxin receptor molecule and the one with the
highest probability of being a true receptor is the maize protein
ABP1, and its equivalents in other species (Hertel 1995, Venis
1253
and Napier 1995, Napier 2001, Warwicker 2001). Other authors have suggested the existence of other extra- or intracellular receptors (Claussen et al. 1996) or that the efflux auxin
carrier is a receptor, as occurs with glucose in yeast cells
(Leyser 2001). Nevertheless, it has not been demonstrated that
ABP1, or any other receptor, plays a role in the formation of
adventitious roots, although the studies of Maurel et al. (1994)
and Leblanc et al. (1999) suggest that the path through which
ABP1 acts may be modulated by the gene rolB, the insertion of
which increases root formation in many species.
An alternative explanation for loss of rooting capacity in
adult material may be associated with the differential expression of genes that affect one or all of the phases in the process.
The action of several rooting-related genes has been studied in
English ivy (Woo et al. 1994), in the rac mutant of tobacco
(Lund et al. 1997) and in loblolly pine (Hutchison et al. 1999).
Based on the same experimental model that we used, Gil et al.
(2003) have reported the isolation and characterization of a
cDNA from oak that is differentially expressed in BS and C
shoots collected at both the end of the multiplication phase and
at the end of the rooting process. The gene (QRCPE) is preferentially expressed in C-derived cultures. Analysis of the hormonal regulation of QRCPE and its possible role in the rooting
process are under study.
Acknowledgments
This study was partially supported by CDI, MCYT, Spain (Project
No. AGL200-0297-CO3-02). G.A. acknowledges the support of a
CONACYT/AECI fellowship during this work.
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