Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:
Tree Physiology 16, 673--680
© 1996 Heron Publishing----Victoria, Canada
Requirements for in vitro rooting of Quercus robur and Q. rubra shoots
derived from mature trees
M. C. SANCHEZ, M. C. SAN-JOSE, A. BALLESTER and A. M. VIEITEZ
Instituto de Investigaciones Agrobiológicas de Galicia (CSIC) Apartado 122, 15080 Santiago de Compostela, Spain
Received February 2, 1996
Summary Stabilized shoot cultures initiated from crown
material of six adult Quercus robur L. trees and from basal
epicormic shoots of a Quercus rubra L. tree showed good in
vitro rooting capacity. An initial five-day dark period generally
improved the rooting response but was detrimental to plantlet
quality. There were clonal differences in rooting capacity. The
concentration and exposure time of the indolebutyric acid
(IBA) treatment were critical for root induction. In both species, best rooting efficiency was achieved by culture in medium
containing 25 mg l −1 IBA for 24 h and subsequent transfer to
an auxin-free medium containing 1% activated charcoal. For
all clones tested, the charcoal benefited both shoot quality and
root system development, the latter being enhanced by the
formation of many lateral roots. Total root system area and
length, measured with a digital image analyzer, were significantly greater in medium containing charcoal than in medium
lacking charcoal. Because darkening the basal part of the
shoots with aluminum foil during the rooting phase only caused
a small increase in rooting, we conclude that the large effect of
charcoal on rooting was the result of adsorption of inhibitory
compounds from the medium or the explant or both, rather than
of basal darkening. Other factors affecting the rooting response
of Q. robur were: (a) the position on the tree of the material
from which cultures were initiated (the topophysical effect);
and (b) shoot quality. Recycling the same horizontally placed
explant on multiplication medium allowed three successive
crops of shoots to be obtained, and rootability was typically
maintained from crop to crop.
Keywords: charcoal, image analysis, micropropagation,
pedunculate oak, red oak.
The establishment and multiplication of shoot cultures derived from mature Quercus rubra L. (red oak) and Q. robur L.
(pedunculate oak) trees have been achieved using explants
isolated from shoots flushed on branch segments (Vieitez et al.
1993, Vieitez et al. 1994) and stem sections (Evers et al. 1993).
The continuous culture of the same horizontal shoot explants
improves the in vitro performance of cultures initiated from
reinvigorated shoots induced by the forced flushing method
(Vieitez et al. 1994). We have observed no decline in shoot
vigor or proliferation capacity after three years of subculturing
with horizontal explants.
Micropropagation requires not only stable shoot multiplication, but also successful rooting of microcuttings. The rooting
of oak shoots of adult origin has received little attention,
probably because of the difficulty in obtaining stabilized shoot
cultures, especially from the crown (Juncker and Favre 1989,
Gebhardt et al. 1993). Acceptable rooting percentages have
been obtained with microshoots initiated from stump sprouts
(Vieitez et al. 1985, Meier-Dinkel 1987, Chalupa 1993) and
from epicormic shoots taken near the base of the trunk (SanJosé et al. 1988, Evers et al. 1993) of adult oak trees. However,
there are few reports on rooting of shoot cultures derived from
the crown. Evers et al. (1993) achieved rooting rates of ≤ 20%
and Vieitez et al. (1994) reported 15--46% rooting rates, but the
regenerated plantlets were of poor quality. No reports have
described in vitro regeneration of plantlets from red oak cultures of mature origin, although Vieitez et al. (1993) achieved
plantlet regeneration with cultures of juvenile origin. In this
study, we investigated the in vitro rooting of mature Q. robur
and Q. rubra. A microcomputer based image analysis technique was used to evaluate root system development.
Introduction
Materials and methods
To exploit genetic gains in forestry species, efficient vegetative
propagation techniques are needed. For the genus Quercus, the
development of both macro- and micropropagation technologies has been suggested (Kleinschmit 1986). However, as with
many woody species, propagation from trees more than 6 to 8
years old is difficult and necessitates rejuvenation (Savill and
Kanowski 1993).
Shoot cultures of Q. robur were initiated from crown branches
(collected at least 5 m from the ground) of six mature trees
aged 70--300 years and designated SL1, SL3, Sainza, Tixon,
Monasterio and San Esteban. Cultures established from Sainza
consisted of three shoot culture lines that were derived from
three sources of explants differing in position of topophysis,
including from basal sprouts (collected less than 0.5 m from
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SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
the ground) and from branch segments taken at 3 and 7 m
above soil level. Tissue culture procedures and conditions were
as previously described (Vieitez et al. 1994). Briefly, accessory
or epicormic shoots were induced on branch segments (3--5 cm
in diameter and 30 cm long) by placing them in a growth
cabinet at 24 °C, in a 16-h photoperiod and a relative humidity
of 80--85%. After 2--3 weeks, flushed shoots (2--10 cm long)
were used as the source of explants for establishing cultures.
Axillary shoots of the six clones were subcultured for 1--2
years before the rooting experiments began.
Quercus rubra source material consisted of epicormic
shoots from the basal zone of the trunk of a 40-year-old tree
designated M1. Epicormic shoots were collected in December,
cut into 25-cm lengths and stored in the cold for 3 months
before being forced to flush in the growth cabinet. The new
growth was used as the source of initial explants.
In both species, clonal shoot multiplication was achieved by
subculturing decapitated shoots placed horizontally in 500-ml
glass jars containing 70 ml of either Gresshoff and Doy (1972)
medium (GD) (for Q. robur) or Woody Plant Medium (WPM)
(Lloyd and McCown 1980) (for Q. rubra). The multiplication
medium was supplemented with 0.2 mg l −1 (0.88 µM) 6-benzyladenine (BA), 30 g l −1 sucrose and 6.5 g l −1 Vitroagar
(Hispanlab S.A.). The pH of the medium was adjusted to
5.5--5.6 before autoclaving at 121 °C and 108 kPa for 20 min.
The multiplication cycle consisted of a 4-week (Q. robur) or
5-week (Q. rubra) period, unless otherwise stated.
Root initiation
For root initiation, shoots 2--3 cm long were excised from
proliferating cultures and their basal halves stripped of leaves.
Rooting medium consisted of the multiplication medium with
reduced macronutrients (GD reduced to one third strength and
WPM reduced by half) and without BA. Unless otherwise
stated, the shoots were cultured six or seven per jar in 300-ml
glass jars containing 60 ml of rooting medium with or without
indole-3-butyric acid (IBA) or activated charcoal (AC, Merck
No. 2186).
Four series of experiments were performed. The first experiment evaluated the efficiency of an initial 5-day dark period
and compared two treatments for initiation of rooting prior to
growth in auxin-free rooting medium: (a) 7 days growth in
rooting medium supplemented with 3 mg l −1 (14.7 µM) IBA,
and (b) dipping the basal end of the shoots in 0.5--1 g l −1
(2.5--4.9 mM) IBA solution for 30 s (Q. robur) or 2 min
(Q. rubra). In the second experiment, shoots were grown for
24 h in rooting medium with 5 to 50 mg l −1 (28.5--245 µM)
IBA before transfer to auxin-free medium. The third series
compared the effects of one month’s growth in auxin-free
medium with or without 1% AC following a 24-h treatment
with IBA at a concentration of 15 mg l −1 (73.5 µM) or 25
mg l −1 (123 µM). The fourth series evaluated in vitro rooting
in a mixture of peat/vermiculite/perlite (1/1/1 by volume).
Following 24 h in 25 mg l −1 IBA medium, shoots were planted
in sterile glass jars containing 100 ml of substrate moistened
with 70 ml of rooting medium containing 20 g l −1 sucrose. In
all of these experiments, cultures were kept in a 16-h photoperiod, except during the 5-day period of darkness of the first
experiment.
Basal darkening
To determine whether the effect of activated charcoal on rooting resulted from basal darkening or from the adsorption of
inhibitory compounds by the charcoal, a set of experiments
was carried out with SL1 and Monasterio shoots treated for
24 h with 25 mg l −1 or 15 mg l −1 IBA, respectively. Basal
darkening was achieved either by including AC in the medium
or by wrapping the culture tube in aluminum foil to the height
of the agar and covering the surface with an opaque
polypropylene disc with two holes for the shoots. Control
shoots were grown in rooting medium without activated charcoal or screening. Test tubes, each containing two shoots in
16 ml of medium, were used. All treated shoots (AC, foil
wrapped and control) were incubated in a 16-h photoperiod.
Recycling of the original explant
An experiment was designed to determine whether continuous
culture (recycling) of the same horizontal explant affected the
rooting response of its new shoots. Once the shoots were
harvested (Cycle 1), the original explant was transferred to
fresh multiplication medium for further reculture (Cycle 2).
The rooting capacity of shoots taken from three consecutive
culture cycles of SL1, SL3 and Monasterio donor shoots cultured in 2-, 3-, 4- or 6-week cycles was evaluated.
Incubation conditions
The shoot multiplication and rooting experiments were conducted in a growth chamber at a day/night temperature of
25/20 °C. An irradiance of 30 µmol m −2 s −1 from cool-white
fluorescent lamps (OSRAM, L 40 W) was provided throughout a 16-h daily photoperiod. An initial 5-day dark period was
applied in the first set of experiments.
Data collection and statistical analysis
At the end of the 1-month rooting period, the percentage of
rooted shoots was assessed. Rooting characteristics of these
shoots were recorded, including the average number of roots
and the length of the longest root. Because shoots rooted in AC
medium produced a large number of secondary and tertiary
roots, an image analyzer was used to quantify the total root
production in terms of projected area and total length of the
root system. Shoot quality was assessed on the basis of apical
necrosis, leaf drop, browning, and senescence.
For each treatment and clone, there were three replicate jars
with 6--7 shoots per jar or 10 replicate tubes with two shoots
per tube (18--21 shoots per treatment). All experiments were
repeated three or four times. The significance of differences
among the rooting results was established by one-way analysis
of variance followed by the Tukey HSD test (Sokal and Rohlf
1981) or LSD test at P < 0.05 to compare means. Arcsine
transformation was applied to percentage data before analysis.
Non-transformed data are presented in tables and figures.
IN VITRO ROOTING OF MATURE OAKS
Results
Quercus robur
In the first series of root initiation experiments, five Q. robur
clones were used (Table 1). An initial five-day dark period
improved rooting frequency in six out of the 11 cases evaluated, but no significant differences were found for number of
roots or longest root length. The two induction treatments
afforded similar rooting frequencies, except for clone SL3,
whose rooting percentage was doubled by combining the 7day IBA treatment with an initial 5-day dark period. The quick
dip method (1 g l −1 IBA for 30 s) generally produced fewer and
shorter roots than the 7-day treatment. Although the quantitative data suggest that 3 mg l −1 IBA plus 5 days of darkness was
the best treatment, the shoots thus treated exhibited browning
and senescence.
Because of the poor results of the first series of experiments,
24-h IBA treatments were tested. The best rooting responses
were obtained with 25 mg l −1 IBA (results not shown). Shoots
treated with 50 mg l −1 IBA exhibited necrosis. The 25 mg l −1
concentration was selected for all clones except Monasterio,
for which 15 mg l −1 was used because of its greater sensitivity.
For all clones except Sainza 3 and Sainza 7, rooting frequencies with the 24-h IBA treatment were higher than those
achieved with a quick dip or 7-day IBA treatment (cf. Table 2
and Table 1). An initial 5-day dark period did not improve the
rooting capacity of SL1 shoots treated with 25 mg l −1 IBA
(results not shown), and was not tried for other clones. In
clones Sainza, San Esteban and SL1 (particularly the latter),
activated charcoal (AC) significantly increased the percentage
of shoots forming roots and increased the root length (Table 2).
The rooting rates of Tixon and SL3 shoots were not signifi-
675
cantly affected by AC, but shoots of all clones grown on AC
medium appeared healthier than those grown without AC,
regardless of whether roots were produced. Activated charcoal
reduced apical necrosis, leaf drop, browning and senescence
and promoted the reinitiation of shoot growth. Generally, the
mean number of roots per rooted shoot was not significantly
altered (except for the Sainza 3 and Sainza 7 clones), but AC
promoted formation of many lateral roots (Figure 1A), and this
was reflected in the total root lengths (Figure 2). In the three
clones evaluated, total root length was significantly greater on
AC medium than on non-AC medium and it also depended on
genotype. Because roots are essentially uniform cylinders, the
projected area followed the same trend as the total root length
(data not shown).
The rooting capacity of Sainza cultures depended on the
location of the original material on the tree. On AC medium,
shoots derived from basal sprouts had a significantly greater
(P < 0.0001) rooting frequency than shoots derived from
branch segments collected 3 or 7 m from the ground. Moreover, rooted shoots of basal origin produced more roots
(P < 0.005) and longer roots (P < 0.0001) than rooted shoots
from higher positions.
Monasterio and SL1 shoots incubated in peat/vermiculite/perlite substrate had rooting rates of less than 10% because
the shoots dried up after a few days despite the addition of
liquid rooting medium.
In the basal darkening experiment, the rooting frequency of
Monasterio shoots was unaffected by either darkening method,
but that of SL1 shoots was significantly increased by both
methods (Table 3). Furthermore, rooting rates of SL1 shoots
were greater in AC medium than in the aluminum foil darkening treatment. The number of roots was not significantly af-
Table 1. Rooting ability of shoot cultures derived from five mature Q. robur trees. Sainza cultures were derived from branch segments collected at
three heights on the tree: basal sprouts (BS), and 3 and 7 m from the ground. Rooting was induced either by dipping in 1 g l − 1 IBA solution for
30 s or by a 7-day treatment in medium supplemented with 3 mg l − 1 IBA. Shoots were cultured with (D) or without (L) an initial period of darkness.
Each value represents the mean (± SE) of at least three experiments with 18 replicates per treatment per clone.
Clone
IBA treatment
(Age, years)
mg l
Monasterio
(80)
−1
Rooting (%)
Number of roots
Longest root length (mm)
Time
L
D
L
D
L
D
3
1000
7 days
30 s
24.2 ± 2.7a
11.1 ± 2.2a
30.1 ± 6.1a
30.5 ± 6.1b
1.2 ± 0.1
1.0 ± 0.1
1.3 ± 0.1
1.5 ± 0.2
13.7 ± 0.1
11.3 ± 6.3
12.3 ± 0.6
15.5 ± 3.1
3
7 days
45.1 ± 3.6a
39.0 ± 3.0a
2.2 ± 0.1
1.8 ± 0.1
36.8 ± 2.5
38.5 ± 2.4
SL1
(100)
3
1000
7 days
30 s
4.1 ± 1.0a
2.1 ± 1.1a
12.9 ± 1.0b
14.7 ± 1.5b
2.5 ± 1.1
1.5 ± 0.4
1.6 ± 0.4
1.6 ± 0.5
22.0 ± 5.0
13.5 ± 1.4
29.4 ± 3.0
26.8 ± 8.8
SL3
(100)
3
1000
7 days
30 s
10.0 ± 3.1a
19.3 ± 4.3a
39.5 ± 5.1b
17.5 ± 9.0a
3.0 ± 1.3
2.3 ± 0.4
2.4 ± 0.4
1.9 ± 2.7
33.0 ± 6.6
19.1 ± 2.7
25.6 ± 7.0
17.9 ± 0.8
3
7 days
16.7 ± 1.7a
33.6 ± 5.5b
2.0 ± 0.3
1.3 ± 0.2
34.7 ± 2.6
29.9 ± 2.5
3
1000
7 days
30 s
0a
8.4 ± 5.9a
21.5 ± 8.1b
17.2 ± 8.4a
0
2.3 ± 0.3
1.5 ± 0.1
1.2 ± 0.1
0
26.7 ± 4.2
23.0 ± 4.5
11.4 ± 1.2
3
7 days
0a
0
0
San Esteban
(100)
Sainza, BS
(300)
Sainza, 3 m
Sainza, 7 m
1
0a
0
Within each row, values of rooting percentage followed by the same letter are not different at P = 0.05, according to the LSD test.
0
676
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
Table 2. Effect of activated charcoal (AC) on the rooting of shoot cultures derived from five mature Q. robur trees. Sainza culture origins are
described in the caption to Table 1. Rooting was induced by 24 h in a medium containing 25 mg l − 1 IBA and subsequent transfer to auxin-free
medium with or without 1% AC for one month. Values represent the mean ± SE of four experiments with 18 replicates per treatment per clone.
Clone
Rooting (%)
Number of roots
Longest root length (mm)
−AC
+AC
−AC
+AC
−AC
+AC
Tixon
63.0 ± 1.6a
64.1 ± 8.4a
1.9 ± 0.3a
2.2 ± 0.4a
51.4 ± 1.4a
47.1 ± 4.4a
Sainza, BS
Sainza, 3 m
Sainza, 7 m
51.9 ± 1.0a
1.4 ± 1.2a
0a
72.6 ± 0.6b
16.7 ± 2.0b
13.0 ± 3.0b
2.1 ± 0.2a
0.3 ± 0.2a
0a
2.7 ± 0.3a
1.3 ± 0.2b
1.3 ± 0.2b
37.8 ± 1.9a
4.5 ± 3.9a
0a
51.7 ± 1.1b
13.4 ± 2.9b
9.6 ± 3.2b
SL3
51.3 ± 0.6a
53.8 ± 3.1a
2.0 ± 0.1a
1.7 ± 0.1a
39.3 ± 1.9a
39.2 ± 1.2a
SL1
2.5 ± 1.3a
66.4 ± 4.8b
1.5 ± 0.4a
1.5 ± 0.1a
20.0 ± 2.1a
48.2 ± 5.5b
63.8 ± 3.0a
85.8 ± 1.5b
2.0 ± 0.2a
2.1 ± 0.1a
42.4 ± 1.5a
59.3 ± 1.4b
San Esteban
1
Within each clone (row), values for a measured variable followed by the same letter are not different at P = 0.05, according to the LSD test.
Figure 1. (A) Root development on
Q. robur shoots (clone SL1) treated with
25 mg l −1 IBA for 24 h and subsequently
incubated for one month in rooting medium with (upper part) and without (lower
part) charcoal. Bar = 1 cm; (B) Rooting of
Q. rubra shoots induced by basal dipping
in 0.5 g l − 1 IBA for 2 min and subsequent
incubation in charcoal-free medium. Scale
in cm; (C) Root development in Q. rubra
induced by culture for 24 h in 25 mg l −1
IBA and subsequent incubation in charcoal-containing medium. The scale is in
cm.
fected by darkening in either clone, but the development of the
root system (in terms of longest root, area and total root length)
was significantly enhanced in AC medium (P < 0.01 for SL1;
P < 0.005 for Monasterio). None of these variables was significantly improved by aluminum wrapping. The quality of both
shoots and roots was also improved in charcoal medium.
Recycling the same horizontal explant increased both the
number of shoots to be rooted and vigor. In all three clones,
acceptable rooting rates were maintained in the second and the
third cycles (Table 4). Rooting frequency among third-cycle
Monasterio shoots was significantly greater than among firstcycle shoots. Within cycles, rooting capacity was affected by
IN VITRO ROOTING OF MATURE OAKS
677
Table 4. Effect of triple recycling of 20 mm donor shoots on the rooting
ability of three mature oak clones. In each reculture, newly developed
shoots were excised, incubated for 24 h in medium containing 25
mg l − 1 IBA, and then transferred to an auxin-free medium containing
activated charcoal. Different reculture periods (2, 3, 4 or 6 weeks)
were tested. Each value represents the mean (± SE) error of four
experiments, each with 18 replicates per treatment per clone.
Reculture #
(Shoot age)
Figure 2. Total lengths of root systems of rooted shoots of three
Q. robur clones. Within each clone there was a significant treatment
effect (P = 0.05) according to the LSD test.
Rooting (%)
Number of
roots
Longest root
length (mm)
Clone SL1
1 (4 weeks)
2 (4 weeks)
3 (4 weeks)
66.4 ± 4.8a
51.3 ± 3.8a
52.5 ± 2.4a
1.5 ± 0.1a
1.4 ± 0.1a
1.7 ± 0.0a
48.2 ± 5.5a
42.7 ± 1.2a
54.2 ± 3.8a
Clone Monasterio
1 (4 weeks)
2 (2 weeks)
2 (3 weeks)
2 (4 weeks)
3 (4 weeks)
52.7 ± 1.6bc
57.4 ± 3.0cd
44.4 ± 2.3ab
38.5 ± 1.0a
64.9 ± 2.3d
1.5 ± 0.0a
1.8 ± 0.0a
1.7 ± 0.2a
1.7 ± 0.1a
1.8 ± 0.1a
49.8 ± 2.9b
29.8 ± 2.5a
32.2 ± 2.2a
38.6 ± 4.5ab
50.6 ± 3.8b
Clone SL3
1 (4 weeks)
1 (6 weeks)
2 (4 weeks)
3 (4 weeks)
16.3 ± 2.7a
53.8 ± 3.1b
57.0 ± 4.5b
43.6 ± 3.8b
1.7 ± 0.5a
1.7 ± 0.1a
2.1 ± 0.2a
1.9 ± 0.2a
33.6 ± 6.8a
39.2 ± 1.2a
48.8 ± 1.5a
36.6 ± 4.6a
1
shoot age, apparently through its relationship to quality. In
clone SL3, in which rooting percentage was significantly
greater for 6-week-old shoots than for 4-week-old shoots in the
first cycle (P < 0.0001), the former were more vigorous and
longer than the 4-week-old shoots. Unlike SL3 shoots, the
rooting frequency of second-cycle Monasterio shoots decreased as shoot age increased.
Quercus rubra
An initial 5-day dark period increased the rooting rates of
shoots regardless of whether the IBA treatment used was a
quick dip (Figure 1B) or a 7-day treatment in 3 mg l −1 IBA
medium, although the increase was statistically significant
only for the quick dip treatment (Table 5). The best rooting
Within each clone and column, values followed by the same letter
are not different at P = 0.05, according to the Tukey HSD test.
percentages were induced by 24-h treatment in 25 mg l −1 IBA
medium (Figure 1C). Subsequent culture on AC medium did
not significantly increase rooting percentages or number of
roots compared with culture on non-AC medium, but the
longest root length was significantly increased. As in the
Q. robur clones, promotion of root branching in the presence
of AC was observed, and shoots rooted in AC medium were
healthier and showed no necrosis.
The rooting capacity of red oak shoots in the mixture substrate did not differ significantly from that achieved in agar
(with or without charcoal), although inadequate contact between shoot and substrate led to shoot desiccation in some
Table 3. Effects of basal darkness, produced by activated charcoal (AC) or aluminum foil wrapping (Al), on in vitro rooting of shoots derived from
crown branches of two mature oak trees. Rooting was induced by culture in IBA-containing medium for 24 h and subsequent transfer to an
auxin-free medium for one month. The projected area and total root length of the root system were measured by digital image processing methods.
Each value represents the mean (± SE) of four experiments with 18 replicates per treatment per clone.
Treatment
IBA
(mg l − 1)
Rooting
(%)
Number of
roots
Longest root
length (mm)
Area
(mm2)
Total root length
(mm)
Clone SL1
Control
Al
AC
25
25
25
11.1 ± 2.0a
23.2 ± 3.3b
55.2 ± 3.5c
1.6 ± 0.1a
1.5 ± 0.1a
1.6 ± 0.1a
30.4 ± 6.0a
25.7 ± 2.5a
48.0 ± 4.6b
55.3 ± 3.7a
68.5 ± 6.7a
135.3 ± 19.0b
49.3 ± 5.3a
59.1 ± 6.6a
116.5 ± 15.6b
Clone Monasterio
Control
Al
AC
15
15
15
29.8 ± 4.8a
32.0 ± 3.6a
33.7 ± 3.2a
1.2 ± 0.1a
1.6 ± 0.1a
1.6 ± 0.2a
6.9 ± 1.1a
9.5 ± 0.5a
29.8 ± 3.7b
30.3 ± 3.8a
31.3 ± 2.8a
66.8 ± 4.6b
23.6 ± 1.7a
25.1 ± 1.4a
66.4 ± 3.0b
1
Within each clone and column, values followed by the same letter are not different at P = 0.05, according to the Tukey HSD test.
678
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
Table 5. Rooting performance of shoots from red oak clone M1 cultured in rooting medium for one month with (D) or without (L) an initial 5-day
dark period. Rooting was induced either by dipping in 0.5 g l − 1 IBA solution for 2 min or by incubation in IBA medium (3, 15 or 25 mg l −1) for
24 h or 7 days. After IBA treatment, shoots were transferred to auxin-free medium with (+AC) or without (−AC) activated charcoal, or to
peat/vermiculite/perlite substrate (S). Each value represents the mean ± standard error of three experiments, each with 18 replicates per treatment.
IBA Treatment
Root development
medium
L
0.5 g l − 1 for 2 min
0.5 g l − 1 for 2 min
3 mg l − 1 for 7 days
3 mg l − 1 for 7 days
15 mg l −1 for 24 h
15 mg l −1 for 24 h
25 mg l −1 for 24 h
25 mg l −1 for 24 h
25 mg l −1 for 24 h
−AC
−AC
−AC
−AC
−AC
+AC
−AC
+AC
S
+
1
D
+
+
+
+
+
+
+
+
Rooting (%)
Number of
roots
Longest root length
(mm)
15.9 ± 1.3a
45.0 ± 6.9cd
10.1 ± 1.4a
17.8 ± 0.6ab
28.9 ± 5.3abc
36.5 ± 4.0bcd
59.7 ± 4.1d
55.8 ± 2.6d
37.3 ± 9.5bcd
1.9 ± 0.1a
2.3 ± 0.5a
1.3 ± 0.2a
1.8 ± 0.2a
1.9 ± 0.1a
2.5 ± 0.3a
2.0 ± 0.2a
2.1 ± 0.4a
1.6 ± 0.2a
18.1 ± 2.0a
25.8 ± 4.9ab
16.3 ± 1.0a
22.9 ± 2.7a
41.3 ± 6.8abc
55.4 ± 8.4bc
39.1 ± 3.8ab
71.9 ± 9.4c
29.7 ± 5.1ab
Within each column, values followed by the same letter are not different at P = 0.05, according to the Tukey HSD test.
cases. Roots developed in the mixture substrate were significantly shorter and thinner than those formed in AC medium
and had a similar appearance to ex vitro formed roots.
Discussion
We have shown that stabilized shoot cultures initiated from
crown material of adult Q. robur and from epicormics of
Q. rubra can be rooted in vitro.
The shoots of many woody plants root better if they are
incubated in darkness during the first phase of rooting and then
transferred to light (McCown 1988). The beneficial effect of
darkness may be ascribed to a dark-induced decrease in peroxidase activity and an increase in endogenous phenol concentrations during root initiation (Druart et al. 1982). Although
darkness stimulates rooting in many species that are difficult
to root in vitro, it generally induces shoot senescence after
7--10 days, thereby decreasing plantlet survival (Rugini et al.
1988). In this study, a 5-day dark period was detrimental to the
quality of the regenerating Q. robur and Q. rubra plantlets,
even when it increased rooting, because it caused shoot necrosis. It is possible that dark-treated shoots may be more
sensitive to the phytotoxic effects of IBA. The dark response
of the San Esteban clone (Table 1) supports Caboni and
Damiano’s (1994) conclusion that dark treatment may not be
required for certain clones or varieties. The concentration of
auxin used to induce rooting and the duration of application
can be critical for rooting response. In our experiments, a 24-h
exposure to 25 mg l −1 IBA (with no dark period) produced the
best rooting frequencies in both species, with the exception of
the Sainza 3 and Sainza 7 clones. Welander (1983) showed that
for apple shoots, when optimum IBA concentrations are used,
the dark treatment has no effect on rooting.
Activated charcoal stimulates rooting of microcuttings in
some woody species (McCown 1988). Normally, AC is included in the root development medium after root-inducing
auxin treatment (Le Roux and Van Staden 1991, Dumas and
Monteuuis 1995). In seedling and 8-year-old Q. robur material, the addition of activated charcoal to the IBA medium
caused a reduction in the percentage of rooted shoots, presumably because of adsorption of the auxin (Evers et al. 1990).
Juncker and Favre (1989) used activated charcoal in the root
expression medium of oak shoots; however, it is not possible
to draw any conclusions about the effect of AC on rooting
capacity from their study because they did not include a control medium lacking AC.
In our study, AC treatment was beneficial for both root
system development and shoot quality. A similar effect on the
number and quality of Pinus pinaster Ait. roots was reported
by Dumas and Monteuuis (1995). The stimulatory effect of
charcoal may involve: (1) the reduction of light intensity at the
base of the shoots, providing an environment conducive to the
accumulation of auxins or cofactors, or both; and (2) the
adsorption of substances such as inhibitory phenolics and any
excess auxin or cytokinin carried over from previous media
(McCown 1988, Bonga and von Aderkas 1992). Darkening the
basal part of olive and almond shoots during the rooting phase
increases their rooting capacity in the same way as an initial
period of darkness (Rugini et al. 1988), and basal shoot darkening has been reported to be essential for in vitro root formation by chestnut and walnut shoots (Rugini et al. 1993).
However, excluding light at the shoot base did not improve the
rooting of Camellia japonica L. (Vieitez et al. 1989) and only
increased the rooting rate of one of three apple cultivars tested
by Karhu and Zimmerman (1993). Nour and Thorpe (1993)
suggest that the mechanism by which activated charcoal improves the growth and quality of cedar shoots is based on its
capacity to adsorb phytohormones, phenolics and other toxins.
In our study, (a) phenolic exudates were observed at the base
of oak shoots when charcoal was not included in the medium
and (b) basal darkening alone, by means of aluminium foil, had
relatively little effect. The positive effect of AC on the in vitro
rooting of oak therefore seems to be a result of the adsorption
of inhibitory compounds from the medium or the removal of
substances from the explants rather than a result of basal
darkening. On the other hand, activated charcoal stimulates
sucrose hydrolysis during autoclaving, which alters the initial
carbon source of the media (Druart and De Wulf 1993). It has
IN VITRO ROOTING OF MATURE OAKS
been suggested that the greater availability of glucose and
fructose in an autoclaved AC medium promotes somatic embryogenesis in maize anther cultures (Büter et al. 1993) and
may also contribute to the observed improvement in rooting of
oak shoots.
The mixture substrate used in this study did not support the
survival of Q. robur shoots, although it does support both
Q. rubra (this work) and chestnut (unpublished results) shoots.
Oak microcuttings of juvenile origin have been successfully
rooted ex vitro in an 80/20 mixture of light peat and perlite
(Meier-Dinkel et al. 1993), which is a more compact substrate
than that used in our experiments. The difference in rooting
ability in vermiculite substrate between apple and walnut has
been related to the basal diameter of the shoots used for rooting
(Navatel and Bourrain 1994). A similar explanation may apply
to our results, because shoot base diameter increased in the
following order: pedunculate oak < red oak < chestnut. Larger
shoot bases are less likely to settle in the interstices between
substrate particles without contacting the substrate, and therefore are less likely to dry up.
Vieitez et al. (1994) reported that recycling the same horizontally placed explant several times increases multiplication
efficiency and allows a shorter culture cycle. We found that the
rooting ability of oak shoot crops taken from successive culture cycles was maintained. However, the best shoot age for
rooting depended on both clone and cycle.
A degree of rejuvenation can sometimes be obtained by in
vitro procedures. Repeated subculturing for shoot multiplication in vitro sometimes brings about rejuvenation, leading to
increased competence for rooting (Zimmerman 1986, Bonga
and von Aderkas 1992). The effect of this process may be a
function of the stabilization period of shoot production. Once
the stabilized state has been attained, rooting potential may be
greater than in the original source tissue, although the expression of this potential depends on both the quality of the microcuttings and the rooting environment (McCown 1988).
Subculturing does not always stimulate the rooting potential of
mature stock. Quercus is one of the genera that McCown and
McCown (1987) characterize as difficult to stabilize and consequently difficult to root. The stabilization of our Quercus
cultures was only achieved after elongated shoots were cultured horizontally on the agar surface (Vieitez et al. 1994). This
procedure acts as a type of layering and stimulates the development of lateral shoots that are elongated and vigorous and
probably in a better physiological condition for root formation.
Thus, subculturing appears to influence rooting capacity of
oak. If rooting in vitro is used as parameter of rejuvenation,
then the rooting rates presented here indicate some degree of
rejuvenation in our Q. robur cultures.
The shoot culture line developed from basal sprouts of the
Sainza tree had higher rooting rates than crown-derived cultures. The position of the material of origin on the tree influenced rooting ability, reflecting a gradient of increasing
maturity from the bottom to the top of the tree. Similar trends
have been observed in chestnut trees (Sánchez and Vieitez
1991) and Sequoia sempervirens (D. Don) Endl. (Bon et al.
1994). The low rootability of Sainza 3 and Sainza 7 material
suggests that in these clones, subculturing failed to achieve the
679
rejuvenation required for successful rooting. However, we note
that Sainza was 300 years old, whereas none of the other trees
was more than 100 years old; this difference in age may
explain why crown material from Sainza was more recalcitrant
to rejuvenation than crown material from the other trees.
Although results achieved in this work do not guarantee the
viability of a large-scale micropropagation of oak, micropropagated plants could be used as stock plants for cutting
propagation. The development of an integrated system would
improve the feasibility of mass propagation of selected oak
clones (Chalupa 1993, Meier-Dinkel et al. 1993).
Acknowledgments
The technical assistance of M. T. Martinez and N. Vidal is acknowledged. This research was partially supported by the Commission of
the European Communities through the ECLAIR programme, contract AGRE 0067 and by DGICYT, contract PB 92-0017.
References
Bon, M.C., F. Riccardi and O. Monteuuis. 1994. Influence of phase
change within a 90-year-old Sequoia sempervirens on its in vitro
organogenic capacity and protein patterns. Trees 8:283--287.
Bonga, J.M. and P. von Aderkas. 1992. In vitro culture of trees. Kluwer
Academic Publishers, Dordrecht, 236 p.
Büter, B., S.M. Pesticelli, R. Berger, J.E. Schmid and P. Stamp. 1993.
Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal. Plant Cell Rep. 13:79--82.
Caboni, E. and C. Damiano. 1994. Rooting in two almond genotypes.
Plant Sci. 96:163--165.
Chalupa, V. 1993. Vegetative propagation of oak (Quercus robur and
Q. petraea) by cutting and tissue culture. Ann. Sci. For. 50, Suppl.
1:295--307.
Druart, P. and O. De Wulf. 1993. Activated charcoal catalyses sucrose
hydrolysis during autoclaving. Plant Cell Tiss. Org. Cult. 32:97--99.
Druart, P., C. Kevers, P. Boxus and T. Gaspar. 1982. In vitro promotion
of root formation by apple shoots through darkness effect on endogenous phenols and peroxidases. Z. Pflanzenphysiol. 108:429--436.
Dumas, E. and O. Monteuuis, 1995. In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants:
influence of activated charcoal. Plant Cell Tiss. Org. Cult. 40:231-235.
Evers, P., E. Vermeer and S. van Eeden. 1990. Genetics and breeding
of oak. Final Report Project MAI BI 0044. EEC Program Wood
Inducing Cork as Renewable Raw Material. Dorschkamps Research Institute for Forestry and Landscape Planning, Wageningen,
The Netherlands.
Evers, P., E. Vermeer and S. van Eeden. 1993. Rejuvenation of Quercus robur. Ann. Sci. For. 50, Suppl. 1:330--335.
Gebhardt, K., U. Frühwacht-Wilms and H. Weisgerber. 1993. Micropropagation and restricted-growth storage of adult oak genotypes.
Ann. Sci. For. 50, Suppl. 1:323--329.
Gresshoff, P.M. and C.H. Doy. 1972. Development and differentiation
of haploid Lycopersicon esculentum. Planta 107:161--170.
Juncker, B. and J. M. Favre. 1989. Clonal effects in propagating oak
trees via in vitro culture. Plant Cell Tiss. Org. Cult. 19:267--276.
Karhu, S.T. and R.H. Zimmermann. 1993. Effect of light and coumarin during root initiation on rooting apple cultivars in vitro. Adv.
Hort. Sci. 7:33--36.
680
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
Kleinschmit, J. 1986. Oak breeding in Germany: experiences and
problems. In Proc. IUFRO Joint Meeting of Working Parties on
Breeding Theory, Progeny Testing and Seed Orchards. Williamsburg, VA, pp 250--258.
Le Roux, J.J. and J. van Staden. 1991. Micropropagation and tissue
culture of Eucalyptus----a review. Tree Physiol. 9:435--477.
Lloyd, G. and B. McCown. 1980. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip
culture. Proc. Int. Plant Prop. Soc. 30:420--427.
McCown, B.H. 1988. Adventitious rooting of tissue cultured plants. In
Adventitious Root Formation in Cuttings. Eds. T.M. Davis, B.H.
Haissig and N. Sankhla. Discorides Press, Portland, OR, pp 289-302.
McCown, D.D. and B.H. McCown. 1987. North American hardwoods. In Cell and Tissue Culture in Forestry, Vol. 3. Eds. J.M.
Bonga and D.J. Durzan. Martinus Nijhoff Publishers, Dordrecht, pp
247--260.
Meier-Dinkel, A. 1987. In vitro Vermehrung und Weiterkultur von
Stieleiche (Quercus robur L.) und Traubeneiche (Quercus petraea
(Matt.) Liebl.). Allg. Forst-u. J. Ztg. 158:199--204.
Meier-Dinkel, A., B. Becker and D. Duckstein. 1993. Micropropagation and ex vitro rooting of several clones of late-flushing Quercus
robur L. Ann. Sci. For. 50, Suppl. 1:319--322.
Navatel, J.C. and L. Bourrain. 1994. Influence of the physical structure
of the medium on in vitro rooting. Adv. Hort. Sci. 8:57--59.
Nour, K.A. and T.A. Thorpe. 1993. In vitro shoot multiplication of
eastern white cedar (Thuja occidentalis). In Vitro Cell. Dev. Biol.
29P:65--71.
Rugini, E., A. Jacoboni and A. Bazzoffia. 1988. A simple in vitro
method to avoid initial dark period and to increase rooting in fruit
trees. Acta Hort. 227:438--440.
Rugini, E., A. Jacoboni and M. Luppino. 1993. Role of basal shoot
darkening and exogenous putrescine treatments on in vitro rooting
and on endogenous polyamine changes in difficult-to-root woody
species. Sci. Hort. 53:63--72.
Sánchez, M.C. and A.M. Vieitez. 1991. In vitro morphogenetic competence of basal sprouts and crown branches of mature chestnut.
Tree Physiol. 8:59--70.
San-José, M.C., A. Ballester and A.M. Vieitez. 1988. Factors affecting
in vitro propagation of Quercus robur L. Tree Physiol. 4:281--290.
Savill, P.S. and P.J. Kanowski. 1993. Tree improvement programs for
European oaks: goals and strategies. Ann. Sci. For. 50, Suppl.
1:368--383.
Sokal. R.R. and F.J. Rohlf. 1981. Biometry. H. Freeman and Company,
New York, 859 p.
Vieitez, A.M., M.C. San-José and A. Ballester. 1989. Progress towards
clonal propagation of Camellia japonica cv ‘Alba Plena’ by tissue
culture techniques. J. Hort. Sci. 64:605--610.
Vieitez, A.M., M.C. San-José and E. Vieitez. 1985. In vitro plantlet
regeneration from juvenile and mature Quercus robur L. J. Hort.
Sci. 60:99--106.
Vieitez, A.M., F. Pintos, M.C. San-José and A. Ballester. 1993. In vitro
shoot proliferation determined by explant orientation of juvenile
and mature Quercus rubra L. Tree Physiol. 12:107--117.
Vieitez, A.M., M.C. Sánchez, J.B. Amo-Marco and A. Ballester. 1994.
Forced flushing of branch segments as a method for obtaining
reactive explants of mature Quercus robur trees for micropropagation. Plant Cell Tiss. Org. Cult. 37:287--295.
Welander, M. 1983. In vitro rooting of the apple rootstock M26 in adult
and juvenile growth phases and acclimatization of the plantlets.
Physiol. Plant. 58:231--238.
Zimmermann, R.H. 1986. Propagation of fruit, nut and vegetable
crops - overview. In Tissue Culture as a Plant Production System for
Horticultural Crops. Eds. R.H. Zimmermann, R.J. Griesbach, F.A.
Hammerschlag and R.H. Lawson. Martinus Nijhoff Publishers,
Dordrecht, pp 183--200.
© 1996 Heron Publishing----Victoria, Canada
Requirements for in vitro rooting of Quercus robur and Q. rubra shoots
derived from mature trees
M. C. SANCHEZ, M. C. SAN-JOSE, A. BALLESTER and A. M. VIEITEZ
Instituto de Investigaciones Agrobiológicas de Galicia (CSIC) Apartado 122, 15080 Santiago de Compostela, Spain
Received February 2, 1996
Summary Stabilized shoot cultures initiated from crown
material of six adult Quercus robur L. trees and from basal
epicormic shoots of a Quercus rubra L. tree showed good in
vitro rooting capacity. An initial five-day dark period generally
improved the rooting response but was detrimental to plantlet
quality. There were clonal differences in rooting capacity. The
concentration and exposure time of the indolebutyric acid
(IBA) treatment were critical for root induction. In both species, best rooting efficiency was achieved by culture in medium
containing 25 mg l −1 IBA for 24 h and subsequent transfer to
an auxin-free medium containing 1% activated charcoal. For
all clones tested, the charcoal benefited both shoot quality and
root system development, the latter being enhanced by the
formation of many lateral roots. Total root system area and
length, measured with a digital image analyzer, were significantly greater in medium containing charcoal than in medium
lacking charcoal. Because darkening the basal part of the
shoots with aluminum foil during the rooting phase only caused
a small increase in rooting, we conclude that the large effect of
charcoal on rooting was the result of adsorption of inhibitory
compounds from the medium or the explant or both, rather than
of basal darkening. Other factors affecting the rooting response
of Q. robur were: (a) the position on the tree of the material
from which cultures were initiated (the topophysical effect);
and (b) shoot quality. Recycling the same horizontally placed
explant on multiplication medium allowed three successive
crops of shoots to be obtained, and rootability was typically
maintained from crop to crop.
Keywords: charcoal, image analysis, micropropagation,
pedunculate oak, red oak.
The establishment and multiplication of shoot cultures derived from mature Quercus rubra L. (red oak) and Q. robur L.
(pedunculate oak) trees have been achieved using explants
isolated from shoots flushed on branch segments (Vieitez et al.
1993, Vieitez et al. 1994) and stem sections (Evers et al. 1993).
The continuous culture of the same horizontal shoot explants
improves the in vitro performance of cultures initiated from
reinvigorated shoots induced by the forced flushing method
(Vieitez et al. 1994). We have observed no decline in shoot
vigor or proliferation capacity after three years of subculturing
with horizontal explants.
Micropropagation requires not only stable shoot multiplication, but also successful rooting of microcuttings. The rooting
of oak shoots of adult origin has received little attention,
probably because of the difficulty in obtaining stabilized shoot
cultures, especially from the crown (Juncker and Favre 1989,
Gebhardt et al. 1993). Acceptable rooting percentages have
been obtained with microshoots initiated from stump sprouts
(Vieitez et al. 1985, Meier-Dinkel 1987, Chalupa 1993) and
from epicormic shoots taken near the base of the trunk (SanJosé et al. 1988, Evers et al. 1993) of adult oak trees. However,
there are few reports on rooting of shoot cultures derived from
the crown. Evers et al. (1993) achieved rooting rates of ≤ 20%
and Vieitez et al. (1994) reported 15--46% rooting rates, but the
regenerated plantlets were of poor quality. No reports have
described in vitro regeneration of plantlets from red oak cultures of mature origin, although Vieitez et al. (1993) achieved
plantlet regeneration with cultures of juvenile origin. In this
study, we investigated the in vitro rooting of mature Q. robur
and Q. rubra. A microcomputer based image analysis technique was used to evaluate root system development.
Introduction
Materials and methods
To exploit genetic gains in forestry species, efficient vegetative
propagation techniques are needed. For the genus Quercus, the
development of both macro- and micropropagation technologies has been suggested (Kleinschmit 1986). However, as with
many woody species, propagation from trees more than 6 to 8
years old is difficult and necessitates rejuvenation (Savill and
Kanowski 1993).
Shoot cultures of Q. robur were initiated from crown branches
(collected at least 5 m from the ground) of six mature trees
aged 70--300 years and designated SL1, SL3, Sainza, Tixon,
Monasterio and San Esteban. Cultures established from Sainza
consisted of three shoot culture lines that were derived from
three sources of explants differing in position of topophysis,
including from basal sprouts (collected less than 0.5 m from
674
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
the ground) and from branch segments taken at 3 and 7 m
above soil level. Tissue culture procedures and conditions were
as previously described (Vieitez et al. 1994). Briefly, accessory
or epicormic shoots were induced on branch segments (3--5 cm
in diameter and 30 cm long) by placing them in a growth
cabinet at 24 °C, in a 16-h photoperiod and a relative humidity
of 80--85%. After 2--3 weeks, flushed shoots (2--10 cm long)
were used as the source of explants for establishing cultures.
Axillary shoots of the six clones were subcultured for 1--2
years before the rooting experiments began.
Quercus rubra source material consisted of epicormic
shoots from the basal zone of the trunk of a 40-year-old tree
designated M1. Epicormic shoots were collected in December,
cut into 25-cm lengths and stored in the cold for 3 months
before being forced to flush in the growth cabinet. The new
growth was used as the source of initial explants.
In both species, clonal shoot multiplication was achieved by
subculturing decapitated shoots placed horizontally in 500-ml
glass jars containing 70 ml of either Gresshoff and Doy (1972)
medium (GD) (for Q. robur) or Woody Plant Medium (WPM)
(Lloyd and McCown 1980) (for Q. rubra). The multiplication
medium was supplemented with 0.2 mg l −1 (0.88 µM) 6-benzyladenine (BA), 30 g l −1 sucrose and 6.5 g l −1 Vitroagar
(Hispanlab S.A.). The pH of the medium was adjusted to
5.5--5.6 before autoclaving at 121 °C and 108 kPa for 20 min.
The multiplication cycle consisted of a 4-week (Q. robur) or
5-week (Q. rubra) period, unless otherwise stated.
Root initiation
For root initiation, shoots 2--3 cm long were excised from
proliferating cultures and their basal halves stripped of leaves.
Rooting medium consisted of the multiplication medium with
reduced macronutrients (GD reduced to one third strength and
WPM reduced by half) and without BA. Unless otherwise
stated, the shoots were cultured six or seven per jar in 300-ml
glass jars containing 60 ml of rooting medium with or without
indole-3-butyric acid (IBA) or activated charcoal (AC, Merck
No. 2186).
Four series of experiments were performed. The first experiment evaluated the efficiency of an initial 5-day dark period
and compared two treatments for initiation of rooting prior to
growth in auxin-free rooting medium: (a) 7 days growth in
rooting medium supplemented with 3 mg l −1 (14.7 µM) IBA,
and (b) dipping the basal end of the shoots in 0.5--1 g l −1
(2.5--4.9 mM) IBA solution for 30 s (Q. robur) or 2 min
(Q. rubra). In the second experiment, shoots were grown for
24 h in rooting medium with 5 to 50 mg l −1 (28.5--245 µM)
IBA before transfer to auxin-free medium. The third series
compared the effects of one month’s growth in auxin-free
medium with or without 1% AC following a 24-h treatment
with IBA at a concentration of 15 mg l −1 (73.5 µM) or 25
mg l −1 (123 µM). The fourth series evaluated in vitro rooting
in a mixture of peat/vermiculite/perlite (1/1/1 by volume).
Following 24 h in 25 mg l −1 IBA medium, shoots were planted
in sterile glass jars containing 100 ml of substrate moistened
with 70 ml of rooting medium containing 20 g l −1 sucrose. In
all of these experiments, cultures were kept in a 16-h photoperiod, except during the 5-day period of darkness of the first
experiment.
Basal darkening
To determine whether the effect of activated charcoal on rooting resulted from basal darkening or from the adsorption of
inhibitory compounds by the charcoal, a set of experiments
was carried out with SL1 and Monasterio shoots treated for
24 h with 25 mg l −1 or 15 mg l −1 IBA, respectively. Basal
darkening was achieved either by including AC in the medium
or by wrapping the culture tube in aluminum foil to the height
of the agar and covering the surface with an opaque
polypropylene disc with two holes for the shoots. Control
shoots were grown in rooting medium without activated charcoal or screening. Test tubes, each containing two shoots in
16 ml of medium, were used. All treated shoots (AC, foil
wrapped and control) were incubated in a 16-h photoperiod.
Recycling of the original explant
An experiment was designed to determine whether continuous
culture (recycling) of the same horizontal explant affected the
rooting response of its new shoots. Once the shoots were
harvested (Cycle 1), the original explant was transferred to
fresh multiplication medium for further reculture (Cycle 2).
The rooting capacity of shoots taken from three consecutive
culture cycles of SL1, SL3 and Monasterio donor shoots cultured in 2-, 3-, 4- or 6-week cycles was evaluated.
Incubation conditions
The shoot multiplication and rooting experiments were conducted in a growth chamber at a day/night temperature of
25/20 °C. An irradiance of 30 µmol m −2 s −1 from cool-white
fluorescent lamps (OSRAM, L 40 W) was provided throughout a 16-h daily photoperiod. An initial 5-day dark period was
applied in the first set of experiments.
Data collection and statistical analysis
At the end of the 1-month rooting period, the percentage of
rooted shoots was assessed. Rooting characteristics of these
shoots were recorded, including the average number of roots
and the length of the longest root. Because shoots rooted in AC
medium produced a large number of secondary and tertiary
roots, an image analyzer was used to quantify the total root
production in terms of projected area and total length of the
root system. Shoot quality was assessed on the basis of apical
necrosis, leaf drop, browning, and senescence.
For each treatment and clone, there were three replicate jars
with 6--7 shoots per jar or 10 replicate tubes with two shoots
per tube (18--21 shoots per treatment). All experiments were
repeated three or four times. The significance of differences
among the rooting results was established by one-way analysis
of variance followed by the Tukey HSD test (Sokal and Rohlf
1981) or LSD test at P < 0.05 to compare means. Arcsine
transformation was applied to percentage data before analysis.
Non-transformed data are presented in tables and figures.
IN VITRO ROOTING OF MATURE OAKS
Results
Quercus robur
In the first series of root initiation experiments, five Q. robur
clones were used (Table 1). An initial five-day dark period
improved rooting frequency in six out of the 11 cases evaluated, but no significant differences were found for number of
roots or longest root length. The two induction treatments
afforded similar rooting frequencies, except for clone SL3,
whose rooting percentage was doubled by combining the 7day IBA treatment with an initial 5-day dark period. The quick
dip method (1 g l −1 IBA for 30 s) generally produced fewer and
shorter roots than the 7-day treatment. Although the quantitative data suggest that 3 mg l −1 IBA plus 5 days of darkness was
the best treatment, the shoots thus treated exhibited browning
and senescence.
Because of the poor results of the first series of experiments,
24-h IBA treatments were tested. The best rooting responses
were obtained with 25 mg l −1 IBA (results not shown). Shoots
treated with 50 mg l −1 IBA exhibited necrosis. The 25 mg l −1
concentration was selected for all clones except Monasterio,
for which 15 mg l −1 was used because of its greater sensitivity.
For all clones except Sainza 3 and Sainza 7, rooting frequencies with the 24-h IBA treatment were higher than those
achieved with a quick dip or 7-day IBA treatment (cf. Table 2
and Table 1). An initial 5-day dark period did not improve the
rooting capacity of SL1 shoots treated with 25 mg l −1 IBA
(results not shown), and was not tried for other clones. In
clones Sainza, San Esteban and SL1 (particularly the latter),
activated charcoal (AC) significantly increased the percentage
of shoots forming roots and increased the root length (Table 2).
The rooting rates of Tixon and SL3 shoots were not signifi-
675
cantly affected by AC, but shoots of all clones grown on AC
medium appeared healthier than those grown without AC,
regardless of whether roots were produced. Activated charcoal
reduced apical necrosis, leaf drop, browning and senescence
and promoted the reinitiation of shoot growth. Generally, the
mean number of roots per rooted shoot was not significantly
altered (except for the Sainza 3 and Sainza 7 clones), but AC
promoted formation of many lateral roots (Figure 1A), and this
was reflected in the total root lengths (Figure 2). In the three
clones evaluated, total root length was significantly greater on
AC medium than on non-AC medium and it also depended on
genotype. Because roots are essentially uniform cylinders, the
projected area followed the same trend as the total root length
(data not shown).
The rooting capacity of Sainza cultures depended on the
location of the original material on the tree. On AC medium,
shoots derived from basal sprouts had a significantly greater
(P < 0.0001) rooting frequency than shoots derived from
branch segments collected 3 or 7 m from the ground. Moreover, rooted shoots of basal origin produced more roots
(P < 0.005) and longer roots (P < 0.0001) than rooted shoots
from higher positions.
Monasterio and SL1 shoots incubated in peat/vermiculite/perlite substrate had rooting rates of less than 10% because
the shoots dried up after a few days despite the addition of
liquid rooting medium.
In the basal darkening experiment, the rooting frequency of
Monasterio shoots was unaffected by either darkening method,
but that of SL1 shoots was significantly increased by both
methods (Table 3). Furthermore, rooting rates of SL1 shoots
were greater in AC medium than in the aluminum foil darkening treatment. The number of roots was not significantly af-
Table 1. Rooting ability of shoot cultures derived from five mature Q. robur trees. Sainza cultures were derived from branch segments collected at
three heights on the tree: basal sprouts (BS), and 3 and 7 m from the ground. Rooting was induced either by dipping in 1 g l − 1 IBA solution for
30 s or by a 7-day treatment in medium supplemented with 3 mg l − 1 IBA. Shoots were cultured with (D) or without (L) an initial period of darkness.
Each value represents the mean (± SE) of at least three experiments with 18 replicates per treatment per clone.
Clone
IBA treatment
(Age, years)
mg l
Monasterio
(80)
−1
Rooting (%)
Number of roots
Longest root length (mm)
Time
L
D
L
D
L
D
3
1000
7 days
30 s
24.2 ± 2.7a
11.1 ± 2.2a
30.1 ± 6.1a
30.5 ± 6.1b
1.2 ± 0.1
1.0 ± 0.1
1.3 ± 0.1
1.5 ± 0.2
13.7 ± 0.1
11.3 ± 6.3
12.3 ± 0.6
15.5 ± 3.1
3
7 days
45.1 ± 3.6a
39.0 ± 3.0a
2.2 ± 0.1
1.8 ± 0.1
36.8 ± 2.5
38.5 ± 2.4
SL1
(100)
3
1000
7 days
30 s
4.1 ± 1.0a
2.1 ± 1.1a
12.9 ± 1.0b
14.7 ± 1.5b
2.5 ± 1.1
1.5 ± 0.4
1.6 ± 0.4
1.6 ± 0.5
22.0 ± 5.0
13.5 ± 1.4
29.4 ± 3.0
26.8 ± 8.8
SL3
(100)
3
1000
7 days
30 s
10.0 ± 3.1a
19.3 ± 4.3a
39.5 ± 5.1b
17.5 ± 9.0a
3.0 ± 1.3
2.3 ± 0.4
2.4 ± 0.4
1.9 ± 2.7
33.0 ± 6.6
19.1 ± 2.7
25.6 ± 7.0
17.9 ± 0.8
3
7 days
16.7 ± 1.7a
33.6 ± 5.5b
2.0 ± 0.3
1.3 ± 0.2
34.7 ± 2.6
29.9 ± 2.5
3
1000
7 days
30 s
0a
8.4 ± 5.9a
21.5 ± 8.1b
17.2 ± 8.4a
0
2.3 ± 0.3
1.5 ± 0.1
1.2 ± 0.1
0
26.7 ± 4.2
23.0 ± 4.5
11.4 ± 1.2
3
7 days
0a
0
0
San Esteban
(100)
Sainza, BS
(300)
Sainza, 3 m
Sainza, 7 m
1
0a
0
Within each row, values of rooting percentage followed by the same letter are not different at P = 0.05, according to the LSD test.
0
676
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
Table 2. Effect of activated charcoal (AC) on the rooting of shoot cultures derived from five mature Q. robur trees. Sainza culture origins are
described in the caption to Table 1. Rooting was induced by 24 h in a medium containing 25 mg l − 1 IBA and subsequent transfer to auxin-free
medium with or without 1% AC for one month. Values represent the mean ± SE of four experiments with 18 replicates per treatment per clone.
Clone
Rooting (%)
Number of roots
Longest root length (mm)
−AC
+AC
−AC
+AC
−AC
+AC
Tixon
63.0 ± 1.6a
64.1 ± 8.4a
1.9 ± 0.3a
2.2 ± 0.4a
51.4 ± 1.4a
47.1 ± 4.4a
Sainza, BS
Sainza, 3 m
Sainza, 7 m
51.9 ± 1.0a
1.4 ± 1.2a
0a
72.6 ± 0.6b
16.7 ± 2.0b
13.0 ± 3.0b
2.1 ± 0.2a
0.3 ± 0.2a
0a
2.7 ± 0.3a
1.3 ± 0.2b
1.3 ± 0.2b
37.8 ± 1.9a
4.5 ± 3.9a
0a
51.7 ± 1.1b
13.4 ± 2.9b
9.6 ± 3.2b
SL3
51.3 ± 0.6a
53.8 ± 3.1a
2.0 ± 0.1a
1.7 ± 0.1a
39.3 ± 1.9a
39.2 ± 1.2a
SL1
2.5 ± 1.3a
66.4 ± 4.8b
1.5 ± 0.4a
1.5 ± 0.1a
20.0 ± 2.1a
48.2 ± 5.5b
63.8 ± 3.0a
85.8 ± 1.5b
2.0 ± 0.2a
2.1 ± 0.1a
42.4 ± 1.5a
59.3 ± 1.4b
San Esteban
1
Within each clone (row), values for a measured variable followed by the same letter are not different at P = 0.05, according to the LSD test.
Figure 1. (A) Root development on
Q. robur shoots (clone SL1) treated with
25 mg l −1 IBA for 24 h and subsequently
incubated for one month in rooting medium with (upper part) and without (lower
part) charcoal. Bar = 1 cm; (B) Rooting of
Q. rubra shoots induced by basal dipping
in 0.5 g l − 1 IBA for 2 min and subsequent
incubation in charcoal-free medium. Scale
in cm; (C) Root development in Q. rubra
induced by culture for 24 h in 25 mg l −1
IBA and subsequent incubation in charcoal-containing medium. The scale is in
cm.
fected by darkening in either clone, but the development of the
root system (in terms of longest root, area and total root length)
was significantly enhanced in AC medium (P < 0.01 for SL1;
P < 0.005 for Monasterio). None of these variables was significantly improved by aluminum wrapping. The quality of both
shoots and roots was also improved in charcoal medium.
Recycling the same horizontal explant increased both the
number of shoots to be rooted and vigor. In all three clones,
acceptable rooting rates were maintained in the second and the
third cycles (Table 4). Rooting frequency among third-cycle
Monasterio shoots was significantly greater than among firstcycle shoots. Within cycles, rooting capacity was affected by
IN VITRO ROOTING OF MATURE OAKS
677
Table 4. Effect of triple recycling of 20 mm donor shoots on the rooting
ability of three mature oak clones. In each reculture, newly developed
shoots were excised, incubated for 24 h in medium containing 25
mg l − 1 IBA, and then transferred to an auxin-free medium containing
activated charcoal. Different reculture periods (2, 3, 4 or 6 weeks)
were tested. Each value represents the mean (± SE) error of four
experiments, each with 18 replicates per treatment per clone.
Reculture #
(Shoot age)
Figure 2. Total lengths of root systems of rooted shoots of three
Q. robur clones. Within each clone there was a significant treatment
effect (P = 0.05) according to the LSD test.
Rooting (%)
Number of
roots
Longest root
length (mm)
Clone SL1
1 (4 weeks)
2 (4 weeks)
3 (4 weeks)
66.4 ± 4.8a
51.3 ± 3.8a
52.5 ± 2.4a
1.5 ± 0.1a
1.4 ± 0.1a
1.7 ± 0.0a
48.2 ± 5.5a
42.7 ± 1.2a
54.2 ± 3.8a
Clone Monasterio
1 (4 weeks)
2 (2 weeks)
2 (3 weeks)
2 (4 weeks)
3 (4 weeks)
52.7 ± 1.6bc
57.4 ± 3.0cd
44.4 ± 2.3ab
38.5 ± 1.0a
64.9 ± 2.3d
1.5 ± 0.0a
1.8 ± 0.0a
1.7 ± 0.2a
1.7 ± 0.1a
1.8 ± 0.1a
49.8 ± 2.9b
29.8 ± 2.5a
32.2 ± 2.2a
38.6 ± 4.5ab
50.6 ± 3.8b
Clone SL3
1 (4 weeks)
1 (6 weeks)
2 (4 weeks)
3 (4 weeks)
16.3 ± 2.7a
53.8 ± 3.1b
57.0 ± 4.5b
43.6 ± 3.8b
1.7 ± 0.5a
1.7 ± 0.1a
2.1 ± 0.2a
1.9 ± 0.2a
33.6 ± 6.8a
39.2 ± 1.2a
48.8 ± 1.5a
36.6 ± 4.6a
1
shoot age, apparently through its relationship to quality. In
clone SL3, in which rooting percentage was significantly
greater for 6-week-old shoots than for 4-week-old shoots in the
first cycle (P < 0.0001), the former were more vigorous and
longer than the 4-week-old shoots. Unlike SL3 shoots, the
rooting frequency of second-cycle Monasterio shoots decreased as shoot age increased.
Quercus rubra
An initial 5-day dark period increased the rooting rates of
shoots regardless of whether the IBA treatment used was a
quick dip (Figure 1B) or a 7-day treatment in 3 mg l −1 IBA
medium, although the increase was statistically significant
only for the quick dip treatment (Table 5). The best rooting
Within each clone and column, values followed by the same letter
are not different at P = 0.05, according to the Tukey HSD test.
percentages were induced by 24-h treatment in 25 mg l −1 IBA
medium (Figure 1C). Subsequent culture on AC medium did
not significantly increase rooting percentages or number of
roots compared with culture on non-AC medium, but the
longest root length was significantly increased. As in the
Q. robur clones, promotion of root branching in the presence
of AC was observed, and shoots rooted in AC medium were
healthier and showed no necrosis.
The rooting capacity of red oak shoots in the mixture substrate did not differ significantly from that achieved in agar
(with or without charcoal), although inadequate contact between shoot and substrate led to shoot desiccation in some
Table 3. Effects of basal darkness, produced by activated charcoal (AC) or aluminum foil wrapping (Al), on in vitro rooting of shoots derived from
crown branches of two mature oak trees. Rooting was induced by culture in IBA-containing medium for 24 h and subsequent transfer to an
auxin-free medium for one month. The projected area and total root length of the root system were measured by digital image processing methods.
Each value represents the mean (± SE) of four experiments with 18 replicates per treatment per clone.
Treatment
IBA
(mg l − 1)
Rooting
(%)
Number of
roots
Longest root
length (mm)
Area
(mm2)
Total root length
(mm)
Clone SL1
Control
Al
AC
25
25
25
11.1 ± 2.0a
23.2 ± 3.3b
55.2 ± 3.5c
1.6 ± 0.1a
1.5 ± 0.1a
1.6 ± 0.1a
30.4 ± 6.0a
25.7 ± 2.5a
48.0 ± 4.6b
55.3 ± 3.7a
68.5 ± 6.7a
135.3 ± 19.0b
49.3 ± 5.3a
59.1 ± 6.6a
116.5 ± 15.6b
Clone Monasterio
Control
Al
AC
15
15
15
29.8 ± 4.8a
32.0 ± 3.6a
33.7 ± 3.2a
1.2 ± 0.1a
1.6 ± 0.1a
1.6 ± 0.2a
6.9 ± 1.1a
9.5 ± 0.5a
29.8 ± 3.7b
30.3 ± 3.8a
31.3 ± 2.8a
66.8 ± 4.6b
23.6 ± 1.7a
25.1 ± 1.4a
66.4 ± 3.0b
1
Within each clone and column, values followed by the same letter are not different at P = 0.05, according to the Tukey HSD test.
678
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
Table 5. Rooting performance of shoots from red oak clone M1 cultured in rooting medium for one month with (D) or without (L) an initial 5-day
dark period. Rooting was induced either by dipping in 0.5 g l − 1 IBA solution for 2 min or by incubation in IBA medium (3, 15 or 25 mg l −1) for
24 h or 7 days. After IBA treatment, shoots were transferred to auxin-free medium with (+AC) or without (−AC) activated charcoal, or to
peat/vermiculite/perlite substrate (S). Each value represents the mean ± standard error of three experiments, each with 18 replicates per treatment.
IBA Treatment
Root development
medium
L
0.5 g l − 1 for 2 min
0.5 g l − 1 for 2 min
3 mg l − 1 for 7 days
3 mg l − 1 for 7 days
15 mg l −1 for 24 h
15 mg l −1 for 24 h
25 mg l −1 for 24 h
25 mg l −1 for 24 h
25 mg l −1 for 24 h
−AC
−AC
−AC
−AC
−AC
+AC
−AC
+AC
S
+
1
D
+
+
+
+
+
+
+
+
Rooting (%)
Number of
roots
Longest root length
(mm)
15.9 ± 1.3a
45.0 ± 6.9cd
10.1 ± 1.4a
17.8 ± 0.6ab
28.9 ± 5.3abc
36.5 ± 4.0bcd
59.7 ± 4.1d
55.8 ± 2.6d
37.3 ± 9.5bcd
1.9 ± 0.1a
2.3 ± 0.5a
1.3 ± 0.2a
1.8 ± 0.2a
1.9 ± 0.1a
2.5 ± 0.3a
2.0 ± 0.2a
2.1 ± 0.4a
1.6 ± 0.2a
18.1 ± 2.0a
25.8 ± 4.9ab
16.3 ± 1.0a
22.9 ± 2.7a
41.3 ± 6.8abc
55.4 ± 8.4bc
39.1 ± 3.8ab
71.9 ± 9.4c
29.7 ± 5.1ab
Within each column, values followed by the same letter are not different at P = 0.05, according to the Tukey HSD test.
cases. Roots developed in the mixture substrate were significantly shorter and thinner than those formed in AC medium
and had a similar appearance to ex vitro formed roots.
Discussion
We have shown that stabilized shoot cultures initiated from
crown material of adult Q. robur and from epicormics of
Q. rubra can be rooted in vitro.
The shoots of many woody plants root better if they are
incubated in darkness during the first phase of rooting and then
transferred to light (McCown 1988). The beneficial effect of
darkness may be ascribed to a dark-induced decrease in peroxidase activity and an increase in endogenous phenol concentrations during root initiation (Druart et al. 1982). Although
darkness stimulates rooting in many species that are difficult
to root in vitro, it generally induces shoot senescence after
7--10 days, thereby decreasing plantlet survival (Rugini et al.
1988). In this study, a 5-day dark period was detrimental to the
quality of the regenerating Q. robur and Q. rubra plantlets,
even when it increased rooting, because it caused shoot necrosis. It is possible that dark-treated shoots may be more
sensitive to the phytotoxic effects of IBA. The dark response
of the San Esteban clone (Table 1) supports Caboni and
Damiano’s (1994) conclusion that dark treatment may not be
required for certain clones or varieties. The concentration of
auxin used to induce rooting and the duration of application
can be critical for rooting response. In our experiments, a 24-h
exposure to 25 mg l −1 IBA (with no dark period) produced the
best rooting frequencies in both species, with the exception of
the Sainza 3 and Sainza 7 clones. Welander (1983) showed that
for apple shoots, when optimum IBA concentrations are used,
the dark treatment has no effect on rooting.
Activated charcoal stimulates rooting of microcuttings in
some woody species (McCown 1988). Normally, AC is included in the root development medium after root-inducing
auxin treatment (Le Roux and Van Staden 1991, Dumas and
Monteuuis 1995). In seedling and 8-year-old Q. robur material, the addition of activated charcoal to the IBA medium
caused a reduction in the percentage of rooted shoots, presumably because of adsorption of the auxin (Evers et al. 1990).
Juncker and Favre (1989) used activated charcoal in the root
expression medium of oak shoots; however, it is not possible
to draw any conclusions about the effect of AC on rooting
capacity from their study because they did not include a control medium lacking AC.
In our study, AC treatment was beneficial for both root
system development and shoot quality. A similar effect on the
number and quality of Pinus pinaster Ait. roots was reported
by Dumas and Monteuuis (1995). The stimulatory effect of
charcoal may involve: (1) the reduction of light intensity at the
base of the shoots, providing an environment conducive to the
accumulation of auxins or cofactors, or both; and (2) the
adsorption of substances such as inhibitory phenolics and any
excess auxin or cytokinin carried over from previous media
(McCown 1988, Bonga and von Aderkas 1992). Darkening the
basal part of olive and almond shoots during the rooting phase
increases their rooting capacity in the same way as an initial
period of darkness (Rugini et al. 1988), and basal shoot darkening has been reported to be essential for in vitro root formation by chestnut and walnut shoots (Rugini et al. 1993).
However, excluding light at the shoot base did not improve the
rooting of Camellia japonica L. (Vieitez et al. 1989) and only
increased the rooting rate of one of three apple cultivars tested
by Karhu and Zimmerman (1993). Nour and Thorpe (1993)
suggest that the mechanism by which activated charcoal improves the growth and quality of cedar shoots is based on its
capacity to adsorb phytohormones, phenolics and other toxins.
In our study, (a) phenolic exudates were observed at the base
of oak shoots when charcoal was not included in the medium
and (b) basal darkening alone, by means of aluminium foil, had
relatively little effect. The positive effect of AC on the in vitro
rooting of oak therefore seems to be a result of the adsorption
of inhibitory compounds from the medium or the removal of
substances from the explants rather than a result of basal
darkening. On the other hand, activated charcoal stimulates
sucrose hydrolysis during autoclaving, which alters the initial
carbon source of the media (Druart and De Wulf 1993). It has
IN VITRO ROOTING OF MATURE OAKS
been suggested that the greater availability of glucose and
fructose in an autoclaved AC medium promotes somatic embryogenesis in maize anther cultures (Büter et al. 1993) and
may also contribute to the observed improvement in rooting of
oak shoots.
The mixture substrate used in this study did not support the
survival of Q. robur shoots, although it does support both
Q. rubra (this work) and chestnut (unpublished results) shoots.
Oak microcuttings of juvenile origin have been successfully
rooted ex vitro in an 80/20 mixture of light peat and perlite
(Meier-Dinkel et al. 1993), which is a more compact substrate
than that used in our experiments. The difference in rooting
ability in vermiculite substrate between apple and walnut has
been related to the basal diameter of the shoots used for rooting
(Navatel and Bourrain 1994). A similar explanation may apply
to our results, because shoot base diameter increased in the
following order: pedunculate oak < red oak < chestnut. Larger
shoot bases are less likely to settle in the interstices between
substrate particles without contacting the substrate, and therefore are less likely to dry up.
Vieitez et al. (1994) reported that recycling the same horizontally placed explant several times increases multiplication
efficiency and allows a shorter culture cycle. We found that the
rooting ability of oak shoot crops taken from successive culture cycles was maintained. However, the best shoot age for
rooting depended on both clone and cycle.
A degree of rejuvenation can sometimes be obtained by in
vitro procedures. Repeated subculturing for shoot multiplication in vitro sometimes brings about rejuvenation, leading to
increased competence for rooting (Zimmerman 1986, Bonga
and von Aderkas 1992). The effect of this process may be a
function of the stabilization period of shoot production. Once
the stabilized state has been attained, rooting potential may be
greater than in the original source tissue, although the expression of this potential depends on both the quality of the microcuttings and the rooting environment (McCown 1988).
Subculturing does not always stimulate the rooting potential of
mature stock. Quercus is one of the genera that McCown and
McCown (1987) characterize as difficult to stabilize and consequently difficult to root. The stabilization of our Quercus
cultures was only achieved after elongated shoots were cultured horizontally on the agar surface (Vieitez et al. 1994). This
procedure acts as a type of layering and stimulates the development of lateral shoots that are elongated and vigorous and
probably in a better physiological condition for root formation.
Thus, subculturing appears to influence rooting capacity of
oak. If rooting in vitro is used as parameter of rejuvenation,
then the rooting rates presented here indicate some degree of
rejuvenation in our Q. robur cultures.
The shoot culture line developed from basal sprouts of the
Sainza tree had higher rooting rates than crown-derived cultures. The position of the material of origin on the tree influenced rooting ability, reflecting a gradient of increasing
maturity from the bottom to the top of the tree. Similar trends
have been observed in chestnut trees (Sánchez and Vieitez
1991) and Sequoia sempervirens (D. Don) Endl. (Bon et al.
1994). The low rootability of Sainza 3 and Sainza 7 material
suggests that in these clones, subculturing failed to achieve the
679
rejuvenation required for successful rooting. However, we note
that Sainza was 300 years old, whereas none of the other trees
was more than 100 years old; this difference in age may
explain why crown material from Sainza was more recalcitrant
to rejuvenation than crown material from the other trees.
Although results achieved in this work do not guarantee the
viability of a large-scale micropropagation of oak, micropropagated plants could be used as stock plants for cutting
propagation. The development of an integrated system would
improve the feasibility of mass propagation of selected oak
clones (Chalupa 1993, Meier-Dinkel et al. 1993).
Acknowledgments
The technical assistance of M. T. Martinez and N. Vidal is acknowledged. This research was partially supported by the Commission of
the European Communities through the ECLAIR programme, contract AGRE 0067 and by DGICYT, contract PB 92-0017.
References
Bon, M.C., F. Riccardi and O. Monteuuis. 1994. Influence of phase
change within a 90-year-old Sequoia sempervirens on its in vitro
organogenic capacity and protein patterns. Trees 8:283--287.
Bonga, J.M. and P. von Aderkas. 1992. In vitro culture of trees. Kluwer
Academic Publishers, Dordrecht, 236 p.
Büter, B., S.M. Pesticelli, R. Berger, J.E. Schmid and P. Stamp. 1993.
Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal. Plant Cell Rep. 13:79--82.
Caboni, E. and C. Damiano. 1994. Rooting in two almond genotypes.
Plant Sci. 96:163--165.
Chalupa, V. 1993. Vegetative propagation of oak (Quercus robur and
Q. petraea) by cutting and tissue culture. Ann. Sci. For. 50, Suppl.
1:295--307.
Druart, P. and O. De Wulf. 1993. Activated charcoal catalyses sucrose
hydrolysis during autoclaving. Plant Cell Tiss. Org. Cult. 32:97--99.
Druart, P., C. Kevers, P. Boxus and T. Gaspar. 1982. In vitro promotion
of root formation by apple shoots through darkness effect on endogenous phenols and peroxidases. Z. Pflanzenphysiol. 108:429--436.
Dumas, E. and O. Monteuuis, 1995. In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants:
influence of activated charcoal. Plant Cell Tiss. Org. Cult. 40:231-235.
Evers, P., E. Vermeer and S. van Eeden. 1990. Genetics and breeding
of oak. Final Report Project MAI BI 0044. EEC Program Wood
Inducing Cork as Renewable Raw Material. Dorschkamps Research Institute for Forestry and Landscape Planning, Wageningen,
The Netherlands.
Evers, P., E. Vermeer and S. van Eeden. 1993. Rejuvenation of Quercus robur. Ann. Sci. For. 50, Suppl. 1:330--335.
Gebhardt, K., U. Frühwacht-Wilms and H. Weisgerber. 1993. Micropropagation and restricted-growth storage of adult oak genotypes.
Ann. Sci. For. 50, Suppl. 1:323--329.
Gresshoff, P.M. and C.H. Doy. 1972. Development and differentiation
of haploid Lycopersicon esculentum. Planta 107:161--170.
Juncker, B. and J. M. Favre. 1989. Clonal effects in propagating oak
trees via in vitro culture. Plant Cell Tiss. Org. Cult. 19:267--276.
Karhu, S.T. and R.H. Zimmermann. 1993. Effect of light and coumarin during root initiation on rooting apple cultivars in vitro. Adv.
Hort. Sci. 7:33--36.
680
SANCHEZ, SAN-JOSE, BALLESTER AND VIEITEZ
Kleinschmit, J. 1986. Oak breeding in Germany: experiences and
problems. In Proc. IUFRO Joint Meeting of Working Parties on
Breeding Theory, Progeny Testing and Seed Orchards. Williamsburg, VA, pp 250--258.
Le Roux, J.J. and J. van Staden. 1991. Micropropagation and tissue
culture of Eucalyptus----a review. Tree Physiol. 9:435--477.
Lloyd, G. and B. McCown. 1980. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip
culture. Proc. Int. Plant Prop. Soc. 30:420--427.
McCown, B.H. 1988. Adventitious rooting of tissue cultured plants. In
Adventitious Root Formation in Cuttings. Eds. T.M. Davis, B.H.
Haissig and N. Sankhla. Discorides Press, Portland, OR, pp 289-302.
McCown, D.D. and B.H. McCown. 1987. North American hardwoods. In Cell and Tissue Culture in Forestry, Vol. 3. Eds. J.M.
Bonga and D.J. Durzan. Martinus Nijhoff Publishers, Dordrecht, pp
247--260.
Meier-Dinkel, A. 1987. In vitro Vermehrung und Weiterkultur von
Stieleiche (Quercus robur L.) und Traubeneiche (Quercus petraea
(Matt.) Liebl.). Allg. Forst-u. J. Ztg. 158:199--204.
Meier-Dinkel, A., B. Becker and D. Duckstein. 1993. Micropropagation and ex vitro rooting of several clones of late-flushing Quercus
robur L. Ann. Sci. For. 50, Suppl. 1:319--322.
Navatel, J.C. and L. Bourrain. 1994. Influence of the physical structure
of the medium on in vitro rooting. Adv. Hort. Sci. 8:57--59.
Nour, K.A. and T.A. Thorpe. 1993. In vitro shoot multiplication of
eastern white cedar (Thuja occidentalis). In Vitro Cell. Dev. Biol.
29P:65--71.
Rugini, E., A. Jacoboni and A. Bazzoffia. 1988. A simple in vitro
method to avoid initial dark period and to increase rooting in fruit
trees. Acta Hort. 227:438--440.
Rugini, E., A. Jacoboni and M. Luppino. 1993. Role of basal shoot
darkening and exogenous putrescine treatments on in vitro rooting
and on endogenous polyamine changes in difficult-to-root woody
species. Sci. Hort. 53:63--72.
Sánchez, M.C. and A.M. Vieitez. 1991. In vitro morphogenetic competence of basal sprouts and crown branches of mature chestnut.
Tree Physiol. 8:59--70.
San-José, M.C., A. Ballester and A.M. Vieitez. 1988. Factors affecting
in vitro propagation of Quercus robur L. Tree Physiol. 4:281--290.
Savill, P.S. and P.J. Kanowski. 1993. Tree improvement programs for
European oaks: goals and strategies. Ann. Sci. For. 50, Suppl.
1:368--383.
Sokal. R.R. and F.J. Rohlf. 1981. Biometry. H. Freeman and Company,
New York, 859 p.
Vieitez, A.M., M.C. San-José and A. Ballester. 1989. Progress towards
clonal propagation of Camellia japonica cv ‘Alba Plena’ by tissue
culture techniques. J. Hort. Sci. 64:605--610.
Vieitez, A.M., M.C. San-José and E. Vieitez. 1985. In vitro plantlet
regeneration from juvenile and mature Quercus robur L. J. Hort.
Sci. 60:99--106.
Vieitez, A.M., F. Pintos, M.C. San-José and A. Ballester. 1993. In vitro
shoot proliferation determined by explant orientation of juvenile
and mature Quercus rubra L. Tree Physiol. 12:107--117.
Vieitez, A.M., M.C. Sánchez, J.B. Amo-Marco and A. Ballester. 1994.
Forced flushing of branch segments as a method for obtaining
reactive explants of mature Quercus robur trees for micropropagation. Plant Cell Tiss. Org. Cult. 37:287--295.
Welander, M. 1983. In vitro rooting of the apple rootstock M26 in adult
and juvenile growth phases and acclimatization of the plantlets.
Physiol. Plant. 58:231--238.
Zimmermann, R.H. 1986. Propagation of fruit, nut and vegetable
crops - overview. In Tissue Culture as a Plant Production System for
Horticultural Crops. Eds. R.H. Zimmermann, R.J. Griesbach, F.A.
Hammerschlag and R.H. Lawson. Martinus Nijhoff Publishers,
Dordrecht, pp 183--200.