Plant Cell, Tissue and Organ Culture (2005) 81:149–158
Springer 2005

Prevention of hyperhydricity in micropropagated carnation shoots by bottom

cooling: implications of oxidative stress

Sadhy Saher, Abel Piqueras, Eladio Hellin & Enrique Olmos*

Centro de Edafologia y Biologia Aplicada del Segura, CSIC, P.O. Box 164, 30100 Murcia, Spain (*requests
for offprints; Fax: +0034-968-39-62-13; E-mail: eolmos@cebas.csic.es)

Received 15 July 2004; accepted in revised form 30 September 2004

Key words: bottom cooling, carnation, hyperhydricity, micropropagation, oxidative stress

Abstract

Carnation shoot cultures were micropropagated in two different agar concentrations (0.58 and 0.85%) and
placed in a bottom cooling system or control conditions. During the culture period of 28 days, it was
observed that relative humidity, hyperhydricity, dry weight, multiplication rate, and the activity of the
antioxidant enzymatic system changed in relation to the agar concentration used and the application of
bottom cooling. The percentage of hyperhydric shoots also showed a significant decrease under bottom
cooling conditions for both agar concentrations. Lipid peroxidation was always lower in shoots cultured
with bottom cooling. All the antioxidant enzymatic activities were lower in bottom cooling treatments
compared to controls. These results show that the normalization of the environmental conditions in vitro
via bottom cooling can prevent the onset of different simultaneous stress reactions concomitant with
hyperhydricity. The present work provides for the first time , direct evidence of a reduced H2O2 generation
in the tissues cultured in bottom cooling able to reduce oxidative stress.

Abbreviations: APX – ascorbate peroxidase; CAT – catalase; DHAr – dehydroascorbate reductase; EDTA –
ethylendiaminetetraacetic sodium salt; GPXs – soluble guaiacol peroxidase; GPXw – cell-wall bound gua-
iacol peroxidase; GR – glutathione reductase; MDA – malondialdehyde; MDHr – monodehydroascorbate
reductase; NADPH – nicotinamide adenine dinucleotide phosphate; PVP – polyvinylpyrrolidone; ROS –
reactive oxygen species; SOD – superoxide dismutase; TBA – thiobarbituric acid; TCA – trichloroacetic acid

Introduction Usually, micropropagation is performed by using

Hyperhydricity is one of the main abnormalities
observed in plant micropropagation. Hyperhydric
shoots exhibit physiological disorders and have
aberrant morphology, characterized by translu-
cent and brittle stems and leaves (Ziv, 1991; Ol-
mos and Hellin, 1998). The occurrence of this
phenomenon is affected mainly by two factors,
the gelling agent of the media and the atmosphere
in the culture vessels (Debergh, 1983; George,
1996; Gribble, 1999; Perez-Tornero et al., 2001).
small-scale vessels with passive ventilation and
poor diffusion of gases between the culture vessel
and the outside atmosphere. To avoid this, vessel
lids with a filter vent of 0.2 lm have been used
(Majada et al., 1997). The use of gas-permeable
membranes has also been reported in combina-
tion with forced ventilation, to modify the
atmosphere of the culture vessel (Yue et al., 1993;
Zobayed et al., 2001). These ventilation systems
were adequate for improvement of micropropa-
gation when hyperhydricity was a problem
150
(Majada et al., 1997, 2000). The major disad-
vantage of increasing ventilation, by whatever
method, is that water is lost from the medium
(George, 1996). A different method for over-
coming hyperhydricity is the bottom cooling
system. Bottom cooling is based on the reduction
of relative humidity of the vessel atmosphere by
condensation on the cooled culture medium
(Vanderschaeghe and Debergh, 1987). Bottom
cooling of the vessels not only reduced the rela-
tive humidity in the containers but also avoided
desiccation of the culture media. This system also
activates transpiration and translocation by
shoots (DeRiek et al., 1997; Piqueras et al., 1998)
and it has been used successfully to recover hy-
perhydric shoots of carnation (Piqueras et al.,
2002) and apricot (Perez-Tornero et al., 2001).
Agar concentration and agar type are known to
influence the growth response of micropropaged
plants (George, 1996; Scholten and Pierik, 1998).
Several authors showed that raising agar concen-
tration overcame hyperhydricity, while drastically
lowering propagation rate (Debergh, 1983; Ziv
et al., 1983). Similarly, hyperhydricity can be
avoided by modifying the types of solidifying
agents (Scholten and Pierik, 1998; Marga et al.,
1997; Perez-Tornero et al., 2001; Whitehouse
et al., 2002).
In a previous paper, we have observed that
micropropagated carnation shoots became hyper-
hydric when grown in 0.58% agar. However, an
increase of agar concentration to 0.85% reduced
hyperhydricity to 15–30%, depending on the
variety. This was accompanied by a decrease of
the oxidative stress in these shoots growing in
0.85% agar (Saher et al., 2004). However, it is still
not known what induces oxidative stress in hy-
perhydric shoots. Oxidative stress triggers cell
death in plants by damaging nucleic acids, protein
and lipids due to ROS. Shoots micropropagated
in vitro are under continuous stress conditions
(Gaspar et al., 2002). It has been proposed that
shoots which are unable to induce their antioxi-
dant defenses (enzymes and soluble reductants)
against activated forms of oxygen will become
hyperhydric (Franck et al., 1998; Saher et al.,
2004).
The aim of the present work was to evaluate the
effect of the combination of bottom cooling system
with different concentrations of agar, with respect
to oxidative stress in hyperhydricity. To this
purpose, shoots grown under bottom cooling have
been compared with those produced in control
vessels.
Material and methods
Plant material and shoot micropropagation
Mother-shoots of three Dianthus caryophyllus
varieties (Oslo, Killer and Alister) were provided by
Barber and Blanc S.A.E. (Puerto Lumbreras,
Murcia, Spain). Shoots were multiplied on MS-
based medium (Murashige and Skoog, 1962), pH
5.8 ± 0.1, supplemented with 3% (w/v) sucrose
and solidified with 0.8% (w/v) agar without plant
growth regulators. Hyperhydricity was induced by
transferring the shoots to the same medium con-
taining 0.58% (w/v) agar. For the cultures, cylindric
glass flasks (15 cm height and diameter 8 cm) closed
with translucent plastic lids were used. A bottom
cooling system was used, as described previously by
Vanderschaege and Debergh (1987). Refrigeration
temperature of the system was 17 C, creating a
temperature gradient of 7 C between the surface
culture medium and the atmosphere. Cultures were
maintained under a 16/8 h light/dark regime. The
culture room temperature was kept at 25 ± 2 C.
Healthy and hyperhydric leaves of carnations were
obtained after 4 weeks of culture.
Multiplication
After 28 days, the total number of shoots pro-
duced from each individual shoot was expressed as
the multiplication rate. Shoot length was measured
after 28 days of culture.
Assays of enzymatic activities
Leaves (1.0 gr FW) were extracted by grinding
with a mortar and pestle, with 50 mM phosphate
buffer (pH 7.8) containing inert sand and 0.3%
PVP, at 2 C. The buffer used for extraction of
ascorbate peroxidase (APX) was supplemented
with 0.2 mM ascorbate. The homogenates were
filtered and centrifuged at 10,000 · g for 15 min.
These fractions were used for spectrophotometric
determinations of all enzymatic activities except
wall-bound peroxidase. For the extraction of this
enzyme, the previous precipitate was resuspended
in the same buffer and washed two times. The final

precipitate was resuspended in the same buffer,
with 1 M KCl and agitated at 4 C for 1 h. After
another centrifugation at 10,000 · g for 15 min,
the new supernatant was used to determine wall-
bound peroxidase. All samples were passed
through Sephadex columns and then desalted.
Total superoxide dismutase (SOD; EC 1.15.1.1)
activity of the samples was determined spectro-
photometrically at 550 nm by the ferricytochrome
c method, using xanthine/xanthine oxidase as the
source of superoxide radicals (McCord and
Fridovich, 1969).
Catalase (CAT; EC 1.11.1.6) was assayed
according to Aebi (1984). The reaction mixture
contained 50 mM potassium phosphate (pH 7.8),
10 mM H2O2 and enzyme extract. Activity was
determined by following the decomposition of
H2O2 at 240 nm. The molar extinction coefficient
was 36 mM)1 cm)1.
Ascorbate peroxidase (APX; EC 1.11.1.11)
activity was determined in a mixture containing
50 mM potassium phosphate (pH 7.0), 1.5 mM
ascorbate, 1.0 mM H2O2 and enzyme extract
(Hodges et al., 1997). Activity was determined
spectrophotometrically by following the H2O2,
dependent decomposition of ascorbate at 265 nm.
Guaiacol peroxidase (GPX; EC 1.11.1.7)
activity was determined in a reaction mixture
composed of 50 mM potassium phosphate buffer
(pH 7.0), 9 mM guaiacol, 10 mM H2O2, and en-
zyme extract (Olmos et al., 1997). The enzyme
activity was measured by monitoring the increase
in absorbance at 470 nm. The molar extinction
coefficient was 26.6 mM)1 cm)1.
Glutathione reductase (GR; EC 1.6.4.2 ) was
assayed by monitoring the glutathione-dependent
oxidation of NADPH. The reaction mixture con-
tained 0.73 ml of 100 mM potassium phosphate
buffer (pH 7.8), 0.1 ml of 100 mM oxidized glu-
tathione (GSSG), 0.1 ml of 15 mM EDTA,
0.02 ml of 10 mM NADPH, and 0.05 ml enzyme
extract (Hodges et al., 1997). Activity was deter-
mined by following the oxidation of NADPH at
340 nm.
Monodehydroascorbate reductase (MDHAR;
EC 1.6.5.4) was determined in a reaction mixture
composed of 0.065 ml of 50 mM Tris–HCl buffer
(pH 7.8), 0.05 ml of 1.25% Triton X-100, 0.1 ml of
0.2 mM NADH, 0.1 ml of 2.5 mM ascorbate,
0.05 ml (12.5 U) of ascorbate oxidase (unit as de-
fined by Sigma Chemical Co.), and 0.05 ml
151
enzyme extract (Hodges et al., 1997). The reaction
was followed by measuring the decrease in absor-
bance at 340 nm, due to NADH oxidation.
Dehydroascorbate reductase (DHAR; EC
1.8.5.4) was determined according to the method
of Dalton et al. (1993). The reaction mixture
contained 0.85 ml of 100 mM potassium phos-
phate (pH 7.8), 0.05 ml of 50 mM reduced gluta-
thione (GSH), 0.05 ml of 4 mM DHA, and
0.05 ml enzyme extract. Activity was determined
by following the reduction of DHA at 265 nm.
Specific activities were expressed as U · mg)1
proteins, where 1 mU ¼ 1 nmol specific substrate
min)1. Protein concentration was determined
according to Lowry et al. (1951), using bovine
serum albumin as a standard.
Spectrofluorimetric determination of H2O2 and
peroxide production
Leaves were cut in small squares (0.5 · 0.5 mm)
and incubated in 5 ml of 50 mM phosphate buffer,
pH 7.8, containing 5 lM 20 ,70 -dichlorofluorescein
diacetate, this nonpolar compound is non-fluo-
rescent but, after deacetylation, is rapidly oxidized
to the highly fluorescent 20 ,70 -dichlorofluorescein
(DCF) by intracellular H2O2 and peroxides. Every
5 min, the incubation media were collected and
measured using a Kontron SFM 25 fluorescence
spectrophotometer with excitation and emission
wavelengths set at 488 and 520 nm, respectively
(Saher et al., 2004). All studies were performed at
least 5 times in separate experiments.
Lipid peroxides determinations
The level of lipid peroxides was determined as
malondialdehyde (MDA) content, by the thio-
barbituric (TBA) reaction as described by Heath
and Packer (1968). One gram of tissue was
homogenized in 5 ml of 0.1% (w/v) trichloroacetic
acid (TCA). The homogenate was centrifuged at
10,000 · g for 5 min and 4 ml of 20% TCA con-
taining 0.5% (w/v) TBA was added to a 1 ml
aliquot of the supernatant. The mixture was
heated at 95 C for 30 min and then quickly
cooled on ice, followed by centrifugation at
10,000 · g for 15 min, the absorbance was then
measured at 532 nm. The concentration of MDA
was calculated using an extinction coefficient of
155 mM)1 cm)1.
152
Results
Relative humidity and micropropagation parameters
Figure 1 shows the evolution of relative humidity
(RH) in the culture vessel of Killer. The atmo-
sphere of vessels with shoots growing on 0.58%
agar and without bottom cooling (CII) shows that
RH was saturated during the 28 days of culture.
However, when these vessels were placed on bot-
tom cooling (BCII), a slight decrease in RH was
observed, that rose again to saturation at the end
of the culture period. Vessels with shoots cultured
in 0.85% agar and without bottom cooling (CI)
had a RH much lower than vessels with 0.58%
agar (CII). In these vessels, RH decreased slightly
during the first 14 days of culture, rising progres-
sively until the end of the culture. When these
vessels were placed on bottom cooling (BCI), RH
evolution showed the lowest percentage values, the
pattern of evolution being similar to vessels with-
out bottom cooling.
Figure 2 shows the effect of bottom cooling
and agar concentration on hyperhydricity
induction in all varieties. Shoots cultured in
0.58% agar without bottom cooling (CII)
developed 80–95% of hyperhydric shoots. How-
ever, for shoots cultured in 0.85% agar with
bottom cooling (BCI) hyperhydricity was fully
prevented. Shoots grown in 0.85% agar without
bottom cooling (CI) and shoots grown with
105
100
95
90
%RH
85
80
75
70
1 3 7 14 21
Days
Figure 1. Relative humidity inside the culture vessels during
culture periods in control 0.85% agar (CI), control 0.58% agar
(CII), 0.85% agar with bottom cooling (BCI) and 0.58% agar
with bottom cooling (BCII) of Killer shoot cultures. Values are
means ± SD (n ¼ 6).
100
a b
a
alister
80 killer
% Hyperhydricity
oslo
60
40
b
a
b
20 a
a
a
0
CI BCI CII BCII
Agar concentration
Figure 2. Hyperhydricity rate of micropropagated carnation
plantlets in control 0.85% agar (CI), control 0.58% agar (CII),
0.85% agar with bottom cooling (BCI), and 0.58% agar with
bottom cooling (BCII) during culture. Values are means ± SD
(n ¼ 40). Significant differences (p < 0.05,) between cultivars
are indicated by different letters.
0.58% agar with bottom cooling (BCII) showed
similar levels of hyperhydricity that remained
between 20–35%.
Figure 3a shows the evolution of dry weight
(DW) during micropropagation. In the three
varieties, DW increased with bottom cooling
compared to the controls with 0.58 or 0.85%
agar. DW was halved in hyperhydric leaves (CII)
compared to the other treatments. Figure 3b
shows the multiplication rate of the three varie-
ties. Multiplication rate only increased signifi-
cantly for Alister grown with bottom cooling,
with 0.85 or 0.58% of agar, compared with
shoots grown without bottom cooling. Alister
and Killer shoots grown in bottom cooling with
0.58% agar (BCII) showed decreased multiplica-
tion rates compared to bottom cooling with
0.85% agar. Figure 3c shows the length of shoots
from the different treatments. Shoots cultured in
0.58% agar without bottom cooling (hyperhydric
BCI
CI
shoots) did not grow. We have observed a sig-
BCII nificant increase in length only in Oslo growing in
CII
0.85% agar with bottom cooling, compared to
28 shoots grown without bottom cooling.
Oxidative stress and antioxidant systems
The time course of H2O2 production is shown in
Figure 4. For this parameter, a similar trend was
observed for the three cultivars. Bottom cooling
reduced H2O2 production to a level similar to the
 153

(a) 16
c c
(hyperhydric shoots) compared to the others.
c
14 b b
b Lipid peroxidation was always lower in shoots
c
c
cultured in bottom cooling (treatment BCI and
12
b BCII), but treatment BCI showed the lowest
10 values.
Tables 2 and 3 show the evolution of antioxi-
% DW

8 a
a
a dant enzymes during shoot development. All the
6
enzymatic activities, except for peroxidases, de-
4 creased at the end of the culture period. The
highest reduction was observed in SOD activity. In
2
Oslo treatments CI and CII, SOD activity was
(b)
0
d 90% less at 28 days compared to 14 days of cul-
7
CI
BCI
ture. All enzymatic activities were always lower in
c
CII bottom cooling treatments. However, treatment
6 BCII
CII (hyperhydric shoots) showed the highest level
b
Multiplication rate

b
5
b for all the enzymatic activities.
4
Both soluble (GPXs) and cell wall-bound gua-
a iacol peroxidases (GPXw) showed a different
3
pattern compared to the other antioxidant
a a a a
2 enzymes. In both cases, the activity was main-
1
tained or increased slightly with time, except in
treatment CII (hyperhydric shoots) where enzyme
(c)
0
activities were increased from 3–5-fold.
c
8 b b
c
7
b

6 c Discussion
c
b
Height (cm)

5
4 application of a bottom cooling system to micro-
b
3 propagated shoot cultures of carnation can be
2 a
a a
1
0
Alister Killer
Figure 3. Dry weight (a), multiplication rate (b), and height (c)
in control 0.85% agar (CI), control 0.58% agar (CII), 0.85%
agar with bottom cooling (BCI), and 0.58% agar with bottom
cooling (BCII) during culture. Values are means ± SD
(n ¼ 50). Significant differences (p < 0.05,) between cultivars
are indicated by different letters.
control with 0.85% agar (CI), however hyperhy-
dric tissues (CII) showed a high increase in fluo-
rescence.
Table 1 shows the evolution of lipid peroxi-
dation during shoot development. In general,
lipid peroxidation increased during the evolution
of shoot culture and showed the same pattern
for all the varieties. The level of lipid per-
oxidation was always higher in treatment CII
According to our present and previous results, the
considered as an effective method for reducing and
preventing hyperhydricity. A temperature reduc-
tion of 3–4 C inside the culture vessel was enough
Oslo
to decrease relative humidity below 90%. Our re-
sults were similar to those reported in Betula pen-
dula and Dianthus sinensis by Böttcher et al.
(1988), where a reduction of only 1 C in temper-
ature lowered relative humidity inside the culture
vessel to below 95%. The same authors reported
that reduced relative humidity, coupled to a rise in
agar concentration, reverted hyperhydricity. This
approach agrees with results from other authors
who observed that a decrease in temperature of 1–
5 C lowered significantly the relative humidity in
the culture vessel (Ghashghaie et al., 1992; Kozai
et al., 1992). It has been reported that the tran-
spiration rate in cultured shoots is almost
proportional to the absolute value of water po-
tential in the atmosphere when the relative
humidity in the culture vessel rises over 90%
(Kozai et al., 1992). Hence, slight differences in
154

180
Alister-14d Killer-14d Oslo-14d
160 CI
BCI
140 CII
BCII
120

100
R.F.U.

80

60

40

20

180
Alister-28d Killer-28d Oslo-28d
160

140

120

100
R.F.U.

80

60

40

20

0 5 10 15 20 25 0 5 10 15 20 25 05 10 15 20 25

Time (min.)

Figure 4. Time course of H2O2 production [Relative fluorescence units (RFU)] in control 0.85% agar (CI), control 0.58% agar (CII),
0.85% agar with bottom cooling (BCI), and 0.58% agar with bottom cooling (BCII) during culture. Values are means ± SD (n ¼ 6).

relative humidity can lead to important differences considerations show a clear relationship between
in the rate of transpiration although absolute hyperhydricity and lack of transpiration. Thus, it
values remain low (Gribble, 1999). The previous is not surprising that slight decreases in relative

Table 1. Percentage of lipid peroxidation (MDA) in control 0.85% agar (CI), control 0.58% agar (CII), 0.85% agar with bottom
cooling (BCI), and 0.58% agar with bottom cooling during culture

Alister Killer Oslo

14d 28d 14d 28d 14d 28d

MDA CI 100b 121.8b 100b 92b 100a 73b

BCI 74.3c 93.4c 62.1d 73.8c 48.7c 25.2d
CII 147.4a 170.9a 127a 114.5a 107.5a 96.6a
BCII 89.8bc 115.6b 70c 98.6ab 67.2b 35.3c

MDA values (nmol · g D W)1) equivalent to 100% were: 57.5 ± 6.2(Alister); 50.5 ± 7.1(Killer); 39.9 ± 1.8(Oslo). Values are
means ± S.D. Significant differences (p < 0.05,) between cultivars are indicated by different letters.
 155

humidity, of around 5%, were able to not only to
reverse but also prevent hyperhydricity, while rel-
ative humidities of 100% raised hyperhydricity to
100%. This could explain our results, where mi-
cropropagated carnation shoots without bottom
cooling and with 0.85% agar in the culture med-
ium (treatment CI) showed a relative humidity of
95% and low hyperhydricity levels, for all three
varieties studied. However, shoots cultured in
0.58% agar without bottom cooling (treatment
CII) showed 100% relative humidity in the culture
atmosphere and the same percentage of hyper-
hydricity. The quality analysis of the cultured
shoots indicated that bottom cooling always in-
creased dry weight, which could be related to a
reduction in water uptake for the shoots micro-
propagated with bottom cooling. As it has been
reported previously, in addition to the reduction of
hyperhydricity, increased agar concentrations can
cause a drastic reduction of the multiplication rate
(Debergh et al., 1981; George, 1996). In our case,
the increase in agar concentration not only
prevented hyperhydricity but also improved the
multiplication rate in the carnation cultivar Alis-
ter. This could be explained by the moderate
concentration of agar (0.85%) used in our exper-
iment, since the inhibitory effects on multiplication
reported previously have been observed when
higher concentrations of agar (1–1.5%) were used
(Debergh, 1983). Similar results have been re-
ported after a reduction of the relative humidity in
the culture atmosphere, by the use of forced ven-
tilation (Majada et al., 1997; Gribble, 1999;
Thomas et al., 2000; Zobayed et al., 2001). Piqu-
eras et al. (1998) have reported a reduction of
hyperhydricity in micropropagated tobacco plants
with bottom cooling, although a reduction in the
multiplication rate was reported.
During the multiplication period studied (14–
28 days) the increase in lipid peroxidation could be
explained by the senescence induced in the cul-
tured shoots during growth (Jimenez et al., 1997).
But the most significant fact was the reduction of
lipid peroxidation in the shoots cultured in bottom
cooling. Bottom cooling also reduced the genera-
tion of H2O2 to levels similar to those of treatment

Table 2. Percentage of antioxidant enzymatic activities (SOD, CAT, GPXs, GPXw) in control 0.85% agar (CI), control 0.58% agar
(CII), 0.85% agar with bottom cooling (BCI), and 0.58% agar with bottom cooling during culture

Alister Killer Oslo

14d 28d 14d 28d 14d 28d

SOD CI 100c 29.8b 100b 27b 100b 10.3b

BCI 42.5d 15.1c 46.3d 10.3c 45.1d 8.9bc
CII 419.4a 72a 190.6a 52.4a 250.7a 24.4a
BCII 117.2b 25.2b 85.4c 21.8b 71.6c 8c
CAT CI 100b 35.9b 100b 55.9b 100 46.3b
BCI 70.5c 23.5c 63.5c 30.6c 76.4 12.3d
CII 456.6a 160.9a 273.1a 213.1a 345.2 166.9a
BCII 99.9b 24.2c 94.4b 46.3b 91.6 26.7c
GPXs CI 100c 112.4b 100b 146.7b 100 52.8b
BCI 115.5b 55.9c 57.3d 53d 38.9 23.1c
CII 521.9a 1484a 449.1a 1697a 528.6 1509a
BCII 92.5c 53.5c 70.9c 73.1c 52.9 57.9b
GPXw CI 100b 98.6b 100b 144.4b 100 161.5b
BCI 42.6c 35.9d 66.1d 75.8d 52.8 53.4d
CII 553.6a 2048a 746a 3916a 896 2933a
BCII 51.7c 55.5c 85.5c 118.5c 68.9 91.3c

SOD (U min)1 mg)1 protein) activities equivalent to 100% were: 32.5 ± 4.8 (Alister); 45.8 ± 7 (Killer); 57.4 ± 4.1 (Oslo).
CAT (U min)1 mg)1 protein) activities equivalent to 100% were: 74.9 ± 15.8 (Alister); 88 ± 4 (Killer); 67.7 ± 10.4 (Oslo).
GPXs(U min)1 mg)1 protein) activities equivalent to 100% were: 243.9 ± 16.7 (Alister); 220.7 ± 21 (Killer); 244,4 ± 29.7 (Oslo).
GPXw(U min)1 mg)1 protein) activities equivalent to 100% were: 20.9 ± 3.2 (Alister); 12.4 ± 1.8 (Killer); 16.1 ± 4 (Oslo). Values
are means ± S.D. Significant differences (p < 0.05,) between cultivars are indicated by different letters.
156

Table 3. Percentage of antioxidant enzymatic activities (APX, MDHr, DHAr, GR) in control 0.85% agar (CI), control 0.58% agar
(CII), 0.85% agar with bottom cooling (BCI), and 0.58% agar with bottom cooling during culture

Alister Killer Oslo

14d 28d 14d 28d 14d 28d

APX CI 100b 51.7b 100b 50b 100b 48.8b

BCI 55d 36c 59.9c 31c 68.4d 32.4c
CII 320.9a 223.2a 259.5a 248.8a 373.8a 182a
BCII 86.3c 42.2bc 71.8c 45.6b 81.6c 43b
MDHr CI 100b 38.9b 100b 67.9b 100b 29.2c
BCI 73.8c 29.1c 74.7c 54.5c 54c 18.5d
CII 313.8a 142.7a 232.1a 211.2a 333a 110.1a
BCII 101.2b 40.2b 88b 67.7b 91.7b 38b
DHAr CI 100b 55.1b 100b 41.1b 100b 23.2b
BCI 69.7c 43c 65.3d 37.6b 60.9d 14c
CII 304.4a 156.1a 178.7a 138a 225a 94.3a
BCII 92.8b 54.9b 86.6c 42.3b 81.3c 28b
GR CI 100b 53.8b 100b 100.4b 100b 45.1c
BCI 74.3c 52.7b 61.3d 59.6d 96b 35.3c
CII 282.8a 229a 193.5a 333a 279.1a 304.9a
BCII 84c 63.1b 85.7c 84.1c 102.9b 61.8b

APX (U min)1 mg)1 protein) activities equivalent to 100% were: 21.1 ± 3.5 (Alister); 25.2 ± 1 (Killer); 24.4 ± 4.6 (Oslo).
MDHr (U min)1 mg)1 protein) activities equivalent to 100% were: 47.8 ± 4 (Alister); 48.3 ± 3.6 (Killer); 60.6 ± 3.9 (Oslo).
DHAr (U min)1 mg)1 protein) activities equivalent to 100% were: 591.5 ± 36 (Alister); 590.5 ± 17.3 (Killer); 667.9 ± 8.7 (Oslo).
GR (U min)1 mg)1 protein) activities equivalent to 100% were: 138.9 ± 20.2 (Alister); 140.2 ± 13.2 (Killer); 162.7 ± 10.2 (Oslo).
Values are means ± S.D. Significant differences (p < 0.05,) between cultivars are indicated by different letters.

CI. In this study, the relation between oxidative
stress and hyperhydricity has been shown directly
by the spectrofluorimetric determination of H2O2
in addition to lipid peroxidation and the response
of the antioxidant enzymatic system which have
been the most frequently studied parameters in
relation to oxidative stress in previous works
(Franck et al., 1995; Olmos et al., 1997; Piqueras
et al., 1998).
The time course of the antioxidant enzymatic
activities showed a reduction with the age of the
cultured shoots. In the case of SOD, it has been
observed that the activity decreases with the aging
of the tissues (Rabinowitch and Fridovich, 1983).
In the same way, it has been reported that young
leaves of tobacco, oat, and pea have more SOD
activity than senescent ones (del Rio et al., 1978;
Dhindsa et al., 1981, 1982). The same has been
observed in senescing carnation petals (Droillard
et al., 1989; Sylvestre et al., 1989; Droillard and
Paulin, 1990). Jimenez et al. (1997) reported a
decrease in the enzymatic activities of the gluta-
thione/ascorbate cycle as well as of ascorbate
peroxidase during senescence in pea leaves with a
concomitant increase in the concentration of
H2O2. A similar process could explain the rise in
lipid peroxidation during the culture period.
In relation to the antioxidant enzymatic activ-
ities studied, a remarkable reduction was observed
in shoots micropropagated with bottom cooling. A
direct relationship exists between SOD activity
induction and the presence of superoxide radicals
(Hassan and Fridovich, 1977). Therefore, the
previous result seems to indicate a reduction in the
generation of superoxide radicals in the tissues
cultured with bottom cooling. However, Piqueras
et al. (1998) have shown that SOD activity in
Nicotiana tabacum micropropagated in bottom
cooling did not show differences, compared to the
control, until day 15 of culture, when an important
reduction was observed. Our results would be in
agreement with the decrease in lipid peroxidation
observed in the shoots cultured with bottom
cooling. The reduction in both ascorbate peroxi-
dase and catalase, H2O2 scavenging enzymes,
would be related to the lower activity of SOD.

Similarly, the reduction in the enzymatic activities
of the gluthatione/ascorbate cycle could be linked
to a reduced need to regenerate ascorbate and
gluthatione, due to decreased stress conditions
(Foyer et al., 1997).
Compared to antioxidant enzymes, both solu-
ble and cell wall peroxidases showed a different
trend. The evolution of both types of peroxidases
was parallel to the hyperhydricity process. The
significant increases in the activities of the perox-
idases observed in hyperhydrated tissues, in addi-
tion to the fact that shoots cultured in treatment
BCI maintained or even slightly reduced peroxi-
dase activities, point towards these enzymatic
activities as a possible source of free radicals. It
has been recently established that peroxidases
could catalyze the formation of hydroxyl in the
presence of a suitable reductant such as NADH
(Chen and Schopfer, 1999; Schweikert et al.,
2002). Our results show that the modifications
imposed by the bottom cooling system on the
abnormal environmental conditions of the cultures
could prevent the onset of different, simultaneous
stress reactions concomitant to hyperhydricity by
a reduction in the level of oxidative stress.
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