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Postharvest Biology and Technology 23 (2001) 133– 142 www.elsevier.

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Preservative solutions containing boric acid delay senescence


of carnation flowers
Marı́a Serrano a,*, Asunción Amorós a, Marı́a Teresa Pretel a,
Marı́a Concepción Martı́nez-Madrid a, Félix Romojaro b
a
Escuela Politécnica Superior, Uni6ersidad Miguel Hernández, Carretera Beniel-Orihuela Km 3.2, 03312 Orihuela (Alicante), Spain
b
Centro de Edafologı́a y Biologı́a Aplicada del Segura (CSIC), Campus de Espinardo, 30100 Murcia, Spain

Received 21 November 2000; accepted 27 February 2001

Abstract

We investigated the effect of a preservative solution containing boric acid on the senescence of cut carnation flowers
(Dianthus caryophyllus L. cv. Master). A 24-h pulse treatment with the preservative solution containing 50, 75 or 100
mM boric acid or continuous treatment with 1 mM boric acid resulted in strong inhibition of the climacteric ethylene
production. Both the pulse and continuous treatments significantly increased flower longevity. Free and conjugated
1-aminocyclopropane-1-carboxylic acid (ACC) and ACC oxidase activity increased in carnation petals during
senescence, although significantly less in boric acid-treated carnations than in control flowers. The levels of putrescine
increased as senescence progressed in both control and boric acid-treated carnations and an increase in spermidine
levels was higher in treated carnations. Abscisic acid levels in petals also increased during senescence, but much less
in boric acid-treated carnations. It is concluded that boric acid prevents the early rise in ethylene production and
considerably improves carnation vase life. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Abscisic acid; Boric acid; Dianthus caryophyllus; Ethylene; Polyamines; Senescence

1. Introduction (ACC synthase) and ACC oxidase (Peiser, 1986;


Serrano et al., 1991), which convert S-adenosyl-
Senescence of carnation flowers is associated methionine (SAM) to ACC and ACC to ethylene,
with a climacteric-like increase in ethylene pro- respectively. SAM is also a precursor for the
duction. Preclimacteric flowers produce a low synthesis of the polyamines spermidine and sper-
constant rate of ethylene. During the climacteric, mine, which are related to young or actively grow-
there is a co-ordinated increase in the activities of ing tissues (Tiburcio et al., 1997).
1-aminocyclopropane-1-carboxylic acid synthase Silver thiosulfate (STS), a known inhibitor of
ethylene action, has become an essential tool for
the delay of senescence of climacteric flowers and
* Corresponding author. Tel.: 34-96-6749616; fax: 34-96-
6749619.
has been applied in the cut flower industry (Reid
E-mail address: m.serrano@umh.es (M. Serrano). and Wu, 1992). However, STS is a potential envi-

0925-5214/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 5 - 5 2 1 4 ( 0 1 ) 0 0 1 0 8 - 9
134 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142

ronmental hazard and many countries currently 2. Materials and methods


prohibit its use. At present, there are very few
alternatives to STS. Some synthetic cyclopropenes 2.1. Plant material
have been shown to bind to the ethylene receptor
and so prevent the physiological action of ethylene Carnations (Dianthus caryophyllus L. cv.
for extended periods. They have, for example, been Master) were obtained from a local greenhouse
shown useful in prolonging petunia (Serek et al., in Puerto Lumbreras (Murcia, Spain) and were
1995) and carnation (Sisler et al., 1996; Sisler and harvested at the commercial opening stage
Serek, 1997) longevity. Ethanol has also been (the petals forming an angle of 120° with the base
shown to be effective in extending the vase life of of the calyx). In the laboratory, the flowers
carnations by inhibiting ethylene production were trimmed to a 15-cm stem length, randomized
(Podd and Van Staden, 1999). and placed individually in test tubes con-
Aminotriazole (AT) is another compound that taining the various treatments. This was consid-
inhibits the climacteric peak of ethylene produc- ered to be day 0 of the experiment. Pulse treat-
tion and prolongs the vase life of carnation flowers ments were performed, holding the carnation
(Altman and Solomos, 1994; Serrano et al., 1999). flowers for 24 h in the preservative solution (PS)
However, AT has been classified as a putative containing 2% sucrose as an energy source,
carcinogen (Sine et al., 1991) and therefore it is Roquat BL-80 (benzalconyl chloride) as a
difficult to use as a cut flower preservative com- microbicide and 25, 50, 75 or 100 mM of boric
acid, in 25 mM citrate buffer pH 4.5. After
mercially. Aminooxoacetic acid (AOA), a known
pulsing, carnation flowers were transferred to dis-
inhibitor of ACC synthase activity (Van Altvorst
tilled water. Flowers pulsed with the PS without
and Bovy, 1995) is currently being used in Holland
boric acid served as controls. In the continuous
as a pulse treatment in the cut flower industry, in
treatment, carnations were maintained in the same
particular for carnations. However, studies with
PS containing 1 mM boric acid until the end of
AOA indicate certain toxicological risks (Wolter-
senescence and flowers maintained in PS without
ing et al., 1987) and it is rather expensive. Boric
boric acid served as controls. The volumes of PS
acid, already included in a mixture of chemicals
or distilled water were maintained at constant
used to improve flower vase life (Odom, 1954), levels by replenishing the test tubes daily.
may be a good competitor as far as price is Each treatment consisted of ten flowers and
concerned. each experiment was repeated twice. Since they
The objective of our research was to investigate gave similar results, the results of only one exper-
the role of a novel preservative solution containing iment are presented. The environmental con-
boric acid in retarding the senescence of carnation ditions maintained throughout the experiment
flowers. This solution was applied in pulse and were temperature 20–22°C, relative humidity
continuous treatments. The levels of ACC and (RH) 75 –80% and a 12 h photoperiod using
some polyamines were also determined in order to white fluorescent light (74.5 mmol s − 1 m − 2). The
establish whether the inhibition of ethylene biosyn- continuous PS plus 1 mM boric acid treatment was
thesis by boric acid would increase polyamine repeated with 40 flowers using another 40
content, which would indicate that both pathways, flowers as control. Every 2 days, three car-
ethylene and polyamine biosynthesis, compete for nations were taken from each treatment (the PS
their common precursor. In addition, we analyzed and the PS plus boric acid). Carnation petals were
the levels of abscisic acid (ABA), a hormone removed from the first, second and third whorls of
related to maturation, especially in non-climacteric the flower and strictly randomized into four
fruit (Serrano et al., 1995; Martı́nez-Madrid et al., groups of 15, nine, six and six petals, in which
1996; Kondo and Inoue, 1997), in order to deter- ACC content, ACC oxidase activity and
mine the role of ABA in controlling carnation polyamine and ABA levels, respectively, were ana-
senescence when ethylene biosynthesis is inhibited. lyzed.
M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142 135

2.2. Ethylene production and respiration rate were cut into small pieces and enclosed in vials
with 25 mM Tris– Hepes buffer, pH 7.5, contain-
To measure ethylene production and respira- ing 1 mM ACC. After 2 h at 30°C and continuous
tion rate, cut carnations were individually en- shaking, a 1-ml gas sample of the vial atmosphere
closed in 500 ml glass jars fitted with a silicon was withdrawn and monitored for its ethylene
septum for 1 h. After this time, a 1 ml sample of content. ACC oxidase activity was expressed as
the jar atmosphere was withdrawn and the ethyl- nanolitres of ethylene released per gram fresh
ene concentration determined using a Hewlett– weight per hour (nl g − 1 h − 1).
Packard model 5890 gas chromatograph
(Wilmington, DE) equipped with a flame ioniza- 2.5. Polyamine extraction and quantification
tion detector (FID) and a 3 m length stainless
steel column with 3.17 mm inner diameter, Polyamines were extracted with HClO4 and an-
filled with 80/100 activated alumina. Results alyzed by the benzoylation method, as previously
were expressed as nanoliters of ethylene produced reported (Serrano et al., 1991). Extracts for
per gram of fresh weight per hour (nl g − 1 h − 1) polyamine analysis were prepared by homogeniz-
and are the mean9S.E. of ten flowers. Another 1 ing six carnation petals in 10 ml of 5% HClO4
ml gas sample of the same jar was used to using a mortar and pestle. The homogenate was
determine CO2 concentration in a Shimadzu 14-B then centrifuged for 30 min at 20 000× g and the
gas chromatograph (Kyoto, Japan). Respiration supernatant was used to quantify the polyamine
rate was expressed as milligram of CO2 re- content in duplicate. A total of 2 ml of the
leased by a kilogram of fresh weight per hour (mg supernatant were mixed with 2 ml of 4 N NaOH
kg − 1 h − 1). Results are the mean9 S.E. of ten and 20 ml of benzoyl chloride in a glass tube.
flowers. After vortexing for 15 s, the mixture was incu-
bated for 20 min at room temperature. Saturated
2.3. ACC extraction and assay NaCl (4 ml) and 4 ml of cold diethyl ether
were then added. The tube content was vortexed
Total (free and conjugated ACC) was extracted for 15 s and incubated for 30 min at − 18°C.
as previously described (Serrano et al., 1991). Finally, 2 ml of the ether phase (containing
Fifteen petals were macerated in a mortar with a benzoyl-polyamines) were evaporated under nitro-
pestle in 10 ml of 0.2 M trichloroacetic acid. The gen and redissolved in 1 ml of methanol (HPLC
macerate was centrifuged at 7000×g for 10 min grade). Benzoyl-polyamines were analyzed by
and the supernatant was used to determine its free HPLC using a Hewlett–Packard system, series
ACC content by chemical conversion of ACC to 1100 (Waldbrom, Germany). The elution
ethylene which was then quantified as described system consisted of methanol:water (64:36, v/v as
above. Conjugated ACC was hydrolyzed to free solvent), run isocratically with a flow rate of 0.8
ACC with 2 N HCl. In both cases, measurements ml min − 1. The benzoyl-polyamines were
were made in triplicate. A relative calibration eluted through a reverse-phase column
procedure was used to determine the amount of (LiChroCart 250-4, 5 mm, Merck, Darmstadt,
ACC in samples using a standard curve of ACC Germany) and detected by absorbance at 254 nm.
from Sigma (Poole, Dorset, UK). Results were A relative calibration procedure was used to de-
expressed as nmol per gram fresh weight (nmol termine the amounts of polyamines in samples
g − 1 f.w.) and are the mean9 S.E. of triplicate using standard curves of putrescine, spermidine
measurements on each of three carnations. and spermine from Sigma and adding hexa-
nediamine as the internal standard. Results were
2.4. ACC oxidase acti6ity expressed as nmol per gram fresh weight (nmol
g − 1 f.w.) and are the mean9S.E. of two mea-
ACC oxidase activity was measured in triplicate surements made independently on three carna-
in each carnation flower. Three carnation petals tions.
136 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142

2.6. Abscisic acid determination PS containing 25 mM boric acid significantly


diminished ethylene production, which reached a
ABA was extracted from six petal samples with maximum of 6.89 1.0 nl g − 1 h − 1, 4 days later
5 ml of a solution of 80% acetone containing 100 than in controls. Pulse treatment with the same
mg l − 1 of butylated hydroxytoluene and 0.5 g l − 1 solution containing 50, 75 and 100 mM of boric
of citric acid. The extracts were diluted with a 50 acid resulted in almost complete inhibition of the
mM Tris buffer, pH 7.8, containing 1 mM MgCl2 climacteric ethylene production (Fig. 1A). The
and 150 mM NaCl and then quantified by an respiration rate also increased during carnation
enzyme-linked immunosorbent assay (ELISA), us- senescence, showing a peak of respiration that
ing an IgG monoclonal antibody (Idetek Inc., San coincided with the peak of ethylene production
Bruno, CA), as previously reported (Martı́nez- (Fig. 1B). The maximum respiration rate of con-
Madrid et al., 1996). ABA content was estimated
trol carnations was significantly higher (PB 0.05)
from the standard curve prepared for each partic-
than in pulse-treated carnations (Fig. 1B).
ular plate using a spectrophotometer StarFax
2100 (Awareness Technology Inc., Palm City,
FL). The absorbance was fixed at 405 nm. For
each extract, four dilutions were made and at least
three fell on the standard curve. All determina-
tions were carried out in dim light. The ABA
levels were consistent with the dilution made and
no interference from impurities was detected when
ABA standards were added to diluted extracts.
Results are expressed as pmol per gram fresh
weight (pmol g − 1 f.w.) and are the mean9S.E.
of the extractions made independently from each
of three flowers and each extract was measured in
quadruplicate.

2.7. Statistics

Experimental data are the mean9S.E. A vari-


ance analysis using Student’s t-test was performed
to determine if treatments showed significant dif-
ferences (PB 0.05).

3. Results and discussion

3.1. Ethylene production and respiration rate

Ethylene production was very low in control


flowers, during the first 7 days at 20°C (Fig. 1A).
Autocatalytic ethylene production started on day
8, reaching a maximum of 15.691.5 nl g − 1 h − 1
Fig. 1. Ethylene production (A) and respiration rate (B) during
on day 12. Maximum ethylene production coin-
senescence of Master carnation flowers at 20°C after pulse
cided with the appearance of visible senescence treatment with PS containing 25, 50, 75 and 100 mM boric
symptoms, such as petal in-rolling and withering. acid. Carnation pulsed with PS without boric acid served as
Pulse treatment of cut carnation flowers with the control. Each value is the mean 9S.E. of ten flowers.
M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142 137

potent inhibitor of ethylene action (Cook and


Van Staden, 1987; Altman and Solomos, 1995)
and suggest that the respiratory climacteric may
be regulated by even very low ethylene levels,
being suppressed only when the ethylene produc-
tion is completely inhibited.

3.2. Flower longe6ity

Flower longevity, defined as the time that car-


nation flowers maintain their decorative proper-
ties (that is, the time until visible symptoms of
senescence appear), was significantly increased by
boric acid treatment. In pulse-treated flowers with
the PS containing boric acid, longevity increased
with the strength of the boric acid concentration
(Table 1). Visible senescence was also delayed in
the continuous treatment with PS plus 1 mM
boric acid (Table 1). In both pulse and continuous
treatments, carnations used as controls were
treated with the PS without boric acid to ensure
that differences between treated and control car-
nations were exclusively due to boric acid. Never-
theless, in a previous experiment we found that
with carnations kept in distilled water, longevity
was slightly shorter than in those treated with the
PS without boric acid, while ethylene production
was similar (data not shown).
Continuous and pulse treatments with PS plus
Fig. 2. Ethylene production (A) and respiration rate (B) during boric acid were also effective in inhibiting ethyl-
senescence at 20°C of continuously-treated carnations with PS
(control) and PS containing 1 mM boric acid. Data are the
ene production and in prolonging flower longevity
mean9 S.E. of ten flowers. in other carnation cultivars, such as Dover,
Eroico, Omagio and Oriana (unpublished data).
Continuous treatment with the PS containing 1 However, no additional increase in longevity was
mM boric acid also drastically inhibited the ethyl- found in carnations continuously treated with the
ene production of carnation flowers (Fig. 2A). PS containing 10 mM boric acid, in which an
undesirable leaf chlorosis was observed, probably
The peak of respiration rate was significantly
due to boric toxicity (unpublished data).
(PB 0.05) lower in the treated flowers and oc-
Until recently, it was assumed that ethylene
curred later (Fig. 2B).
production and petal in-rolling in carnation were
Thus, a peak of respiration was observed in all
regulated in concert, since these phenomena coin-
carnation flowers treated with boric acid (Fig. 1B cided. However, we found that petal in-rolling
and Fig. 2B). However, boric acid treatments was suppressed in carnations flowers pulsed with
(except in the pulse with PS containing 25 mM the PS containing 75 and 100 mM boric acid, as
boric acid) virtually prevented the climacteric well as in those kept continuously in PS plus 1
peak of ethylene production (Fig. 1A and Fig. mM boric acid, while it was observed in those
2A). These results are in agreement with those pulsed with a lower concentration (50 mM) of
found in carnation flowers treated with STS, a boric acid, which produced a similar low level of
138 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142

ethylene. These findings are consistent with the on day 12 (Fig. 3B). However, in carnation flow-
proposal that petal in-rolling and withering are ers kept continuously in PS with 1 mM boric acid,
ethylene independent. They agree with the results ACC oxidase remained very low, only a small
of Satoh et al. (2000) and Onoue et al. (2000), increase being observed on day 16 (Fig. 3B).
who reported that the expression of ACC syn- These results show that the lack of ethylene pro-
thase and ACC oxidase genes and that of cysteine duction in boric acid-treated carnations could be
proteinase (related to withering of petals) were due to a combination of two causes: a low
regulated differently in carnation petals. availability of free ACC, probably resulting from
The continuous treatment with the PS contain- low ACC synthase activity, and the failure to
ing 1 mM boric acid was selected to study the convert ACC into ethylene because of decreased
effect of boric acid on ethylene, polyamines and ACC oxidase activity.
ABA biosynthesis. In untreated carnations, the ability of petal
tissue to convert exogenous ACC into ethylene in
3.3. ACC le6els and ACC oxidase acti6ity the presence of boric acid was assayed at two
developmental stages. Ethylene production in-
Free and total ACC levels were very low in creased with increasing ACC concentrations in
recently harvested flowers and rose slowly in con- both preclimacteric and climacteric carnations
trol flowers during the first 8 days of senescence at (Table 2). The higher ethylene production rate in
20°C (Fig. 3A). After day 8, free and total ACC climacteric petals shows that ACC oxidase activ-
levels rose sharply (Fig. 3A). In carnations main- ity increased during senescence (as can also be
tained in the PS with 1 mM boric acid, free and inferred from Fig. 3B). However, no significant
total ACC levels in the petals were significantly differences in ethylene production were found
lower than in control flowers (Fig. 3A). Although when 10 and 20 mM boric acid were added to the
ACC synthase was not directly measured, results incubation medium. This finding shows that boric
show that boric acid treatment probably inhibited acid does not directly inhibit ACC oxidase activ-
this enzyme by \50%, but did not inhibit ACC ity, as do cobalt ions and a-aminoisobutyric acid
conjugation, since only a small fraction of the (Serrano et al., 1990). Thus, the low ACC oxidase
ACC synthesized remained in a free state (Fig. activity and ACC levels found in boric acid-
3A). treated carnation petals during senescence may be
ACC oxidase activity in control carnations was due to the effect of boric acid on the synthesis of
very low during preclimacteric stages but in- both ACC synthase and ACC oxidase, which
creased along with ethylene production, peaking normally occurs during carnation senescence

Table 1
Longevity (days) of Master carnation flowers after pulse treatment with a pretreatment solution containing various concentrations
of boric acid or continuously kept in the solution containing 1 mM boric acida

Pulse treatments (boric acid (mM) concentration in the PS)

0 25 50 75 100

Longevity 12.2 90.4a 16.79 1.0b 20.8 9 0.7c 21.7 9 0.7c 22.29 0.5c
Continuous treatment (boric acid (mM) concentration in the PS)

0 1

Longevity 11.8 90.6a 18.59 90.7b

a
Each experiment consisted of ten flowers. Means within each treatment group followed by different letters are significantly
different (PB0.05).
M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142 139

the first 8 days of senescence and from day 8 they


increased sharply (Fig. 4A). In boric acid-treated
carnations putrescine also increased during senes-
cence, although slowly, reaching a final value
which was not significantly different from the
levels in controls (Fig. 4A).
Spermidine content was significantly higher
than the putrescine content (Fig. 4A,B), and de-
creased until day 10 in both control and treated
carnation petals. Then, spermidine content in-
creased again until the end of senescence. A simi-
lar pattern of spermidine behaviour has been
reported in other carnation cultivars (Roberts et
al., 1984; Serrano et al., 1991). In carnations
treated with the PS plus 1 mM boric acid, sper-
midine levels increased more than in controls
(Fig. 4B).
Thus, taking into account the antisenescent
properties of the polyamines spermidine and sper-
mine (Tiburcio et al., 1997), the high spermidine
levels could be responsible for the greater
longevity of the boric acid-treated carnations.
However, the rise in spermidine seems to come
too late to account for the delay in the ethylene
rise. The sharp increase in spermidine levels in the
petals of boric acid-treated carnation (in which

Fig. 3. Free and total ACC content (A) and ACC oxidase
activity (B) evolution in petals from continuously-treated car- Table 2
nations with PS (control) and PS containing 1 mM boric acid, Ethylene production (nl g−1 h−1) of carnations petals at two
during senescence at 20°C. Data are the mean 9 S.E. of mea- stages of development after incubation for 2 h with various
surements made in triplicate in each of three flowers. ACC concentrations plus 10 or 20 mM boric acida

(Jones and Woodson 1999; Satoh et al., 2000). ACC Boric acid concentration (mM)
Another possibility is that the low ACC oxidase concentration
(mM) 0 10 20
activity could be due to substrate limitation (Fig.
3). Since ACC synthase and ACC oxidase activity Preclimacteric stage
are in an autocatalytic loop, the present data do 0.1 22.3 9 3.7a 19.1 93.1a 25.4 9 3.2a
not suggest which part of the chain is broken. 0.5 157.4 911.6b 132.6 912.9b 149.6 914.8b
1 258.1 912.9c 282.7 925.0c 294.0 921.5c
Climacteric stage
3.4. Polyamine le6els 0.1 229.9 919.2a 226.5 926.8a 204.0 919.0a
0.5 267.2 98.7b 285.4 922.2b 269.1 921.0b
The most abundant polyamines in the carna- 1 367.8 924.3 386.5 925.7c
c
361.1 917.0c
tion petals of the cultivar Master were putrescine a
Data are the mean 9S.E. of two replicates of three petal
and spermidine. Spermine levels were very low, samples per treatment. Means within each row and column
just at or below the detection limit. In control (for each development stage) followed by different letters are
flowers, putrescine levels increased slowly during significantly different (PB0.05).
140 M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142

previously (Eze et al., 1986; Hanley and Bram-


lage, 1989). Application of exogenous ABA to
carnation flowers (through the cut stem end)
caused a rapid increase in the ABA content in
flower tissues and promoted ethylene production
in the flowers (Onoue et al., 2000). These findings
are in agreement with the proposal that ABA may
play a role in the induction of ethylene production
during natural senescence in carnation flowers.
In boric-acid treated carnations (Fig. 5), as well
as in those treated with AT (Serrano et al., 1999),
the ABA content increased to half of the level
reached in control carnations, whereas the ethyl-
ene production of treated carnations was very
low, which might indicate a separation of the
relationship between these two hormones. In ad-
dition, the ABA levels reached in boric acid-
treated carnations were lower than that in control
flowers, which may point to an inhibition of ABA
biosynthesis by boric acid either directly or by
some intermediate effect.

4. Conclusions

We conclude that a pulse or continuous treat-


ment with a preservative solution containing boric
acid was effective in prolonging carnation flower
Fig. 4. Putrescine (A) and spermidine (B) levels in petals of
continuously-treated carnations with PS (control) and PS con-
taining 1 mM boric acid, during senescence at 20°C. Data are
the mean 9S.E. of quantification made in duplicate in each of
three flowers.

ethylene production was inhibited) may also indi-


cate competition between the ethylene and
polyamine pathways for their common precursor.

3.5. ABA le6els

The ABA content in freshly harvested carna-


tions was low and levels accumulated from day 6
onwards (Fig. 5). In carnations continuously
treated with the PS containing 1 mM boric acid,
ABA started to increase from day 12 (Fig. 5) and
Fig. 5. Evolution of petal ABA content during senescence at
reached values significantly lower than those in 20°C of continuously-treated carnations with PS (control) and
control flowers. ABA thus accumulated before the PS containing 1 mM boric acid. Data are the mean 9S.E. of
onset of ethylene production, as has been reported measurements made in quadruplicate in each of three flowers.
M. Serrano et al. / Posthar6est Biology and Technology 23 (2001) 133–142 141

longevity and could be useful in the cut flower production in tomato fruit. Phytochemistry 43, 323 –
industry. Boric acid treatments apparently inhib- 326.
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