Vol.2, No.4, 498-504 (2011)
doi:10.4236/as.2011.24064
C
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Agricultural Scienc es
Changes of phenolic compounds in Carignan
merithallus (Vitis vinifera L.) during bud dormancy and
end of dormancy phase: correlation with rhizogenesis
Kraiem Dardour Zohra1*, Zairi Asma1, Msaada Kamel2, Hamdi Helmi3, Ezzili Béchir1
1Biotechnology Center in Borj Cedria Technopole, Hammam-lif, T unisia; *Corresponding Author:
2Laboratory of Bioactive Substances, Biotechnology Center in Borj Cedria Technopole, Hammam-lif, T unisia;
3Water Research and Technology Center, Borj Cédria Technopole, Soliman, Tunisia.
Received 29 August 2011; revised 17 September 2011; accepted 21 October 2011.
ABSTRACT
The aim of the present study is to address the
type of correlation that may exist between phe-
nolic compounds and vine rhizogenetic poten-
tial by analyzing some phenolic compounds in
the Carignan merithallus. Phenolic compounds
were analyzed by HPLC in the young shoots (or
merithallus) of Carignan vine (Vitis vinifera L.)
and we established a correlation between the
studied compounds and the rhizogenetic po-
tential of shoots during the phases of bud dor-
mancy and end of dormancy, taking into ac-
count the position effect on shoots. This work
was carried out for the first time on this type of
vine. Among the studied phenolic compounds,
we observed a negative correlation between
coumarin and rhizogenetic potential of Carignan
vine. In contrast, positive correlations were found
with naringin and syringic acid. Obtained re-
sults confirmed the fact that the action of phe-
nolic compounds is complex and might be
qualified as cofactors th at interact with auxin on
rhizogenesis.
Keywords: Phenolic Compounds; HPLC; Carignan
Shoots; Rhizogenesis
1. INTRODUCTION
Polyphenols, a group of substances with a broad spec-
trum of physiological activities, are spread in plants used
in traditional and modern medical systems. Phenolics
have been shown to have a role in tissue browning, fla-
vor, and color characteristics of the derived products [1].
An understanding of phenolic composition in fresh fruit
and the factors that affect phenolic compounds are criti-
cal in the design of products and storage conditions [2].
Many studies have been undertaken on the phenolic acid
compositions of various fruits and their related cultivars
such as apples and pears [3,4], Pyrus [5,6], pome and stone
fruit [7], Diospyros [8], carrots [9], and Prunus [10].
Phenolic compounds are active biological molecules
having one or more benzene rings with one or more hy-
droxyl functions [11]. At the plant level, phenolic com-
pounds contribute to development, cell multiplication,
reproduction, differen tiation, flowering, and lignification.
Their content in plants depends on many genetic, phy-
siological and environmental parameters [12].
Phenolic compounds accumulate mainly in the cell
membrane (lignin and some flavonoids) and in vacuoles
where soluble phenols such as chlorogenic acid, antho-
cyanins, flavonols and tannins are stocked [13]. These
compounds can participate also in the phenomenon of
rhizogenesis. Some authors reported that such contribu-
tion might be positive [14-17]. However, other studies
showed no or negative effects of these compounds on
rhizogen esi s [1 8 - 2 1].
The purpose of this work was to establish the correla-
tion type that may exist between phenolic compounds
and vine rhizogenetic potential by analyzing some phe-
nolic compounds in the Carignan merithallus.
2. EXPERIMENT
2.1. Plant Material
The influence of the sampling date on the rhizogenetic
potentiality of vine merithallus has been previously
studied [22], Carignan vines grown in double Guyot at
the experimental station of the Obligatory Grouping of
Wine Growers and Fruit Producers (GOVPF) in Baddar
(Northeast Tunisia). In this study, vine shoots of various
Carignan genotypes were sampled monthly on five spe-
cific dates that coincide with the phenomenon of bud
dormancy and end of dormancy [23]. The first sampling

K. D. Zohra et al. / Agricultural Sciences 2 (2011) 497-504
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499499
took place on September 13, 2005, the second on Octo-
ber 13, 2005, the th ird on November 13 , 2005, th e fourth
on December 13, 2005 and the last on January 13, 2006.
Thirty merithallus of Carignan vine were sampled
from various rows and positions according the conven-
tion of friml [24]. Samples were dried and reduced to a
fine powder prior to phenolic compound analysis.
2.2. Polyphenols Analysis
2.2.1. Determination of Total Phenolic
Compounds
Analysis of total polyphenols in Carignan merithallus
samples was carried out using Folin-Ciocalteu reagent
[25]. In alkaline medium, this reagent is reduced to
tungsten and molybdenum oxides, giving a blue color in
the presence of polyphenols. Briefly, 125 µL of diluted
extract (methanol 80%) was mixed with distilled water
(500 µL) and 125 µL of Folin-Ciocalteu reagent. Then,
the mixture was agitated, paused for 3 min before the
addition of 1250 µL of CO3(Na)2 at 7%. Finally, the
mixture’s volume was adjusted by distilled water to 3
mL. After 90 min in darkness, absorbance was deter-
mined at a wavelength of 760 nm. The standard curve
was prepared using gallic acid at 50, 100, 200, 3 00, 400,
and 500 mg/L. Total polyphenol content was expressed
as mg of gallic acid equivalent p er g dw.
2.2.2. Solvent Extraction
Solvent extraction was performed according to the
method described by [26]. Merithallus sample (2.5 g dw)
was mixed with 15 mL water, agitated for 30 min and
kept for 24 h at 4˚C in total darkness. The mixture was
then filtered on ash-free filter paper (Whatman n 4) be-
fore storage at 4˚C for subsequent analyses.
2.2.3. HPLC Analysis
Phenolic compounds were identified and quantified
using RP-HPLC coupled to a UV-visible detector and
equipped with a specific C18 column: Hypersil ODS (250
× 4.6 mm, 4 µm). The mobile phase consisted of ace-
tonitrile (solvent A); and a mixture of HPLC-grade water
and sulphuric acid at 0.2% (solvent B). The gradient
program was set up for the mixture (A)-(B) as follows:
15% - 85% for 0 - 12 min, 40% - 60% for 12 - 14 min,
60% - 40% for 14 - 18 min, 80% - 20% for 18 - 20 min,
90% - 10% for 20 - 24 min, and 100% - 0% for the last
24 - 28 min. Injected volume was 20 µL and peaks were
examined at 280 nm. Peaks were characterized by their
retention times and corresponding compounds were id-
entified as compared to retention times of pure stan-
dards. All chemical analyses were conducted in tripli-
cate.
2.3. Statistical Analysis
All determinations were performed in triplicate and
the results are expressed as means values ± standard
deviations (SD). The data were subjected to statistical
analysis using statistical program package STATISTICA
[27]. The one-way analysis of variance (ANOVA) fol-
lowed by Duncan multiple range test were employed and
the differences between individual means were deemed
to be significant at P < 0.05. Correlation coefficients were
calculated based on coumarin, naringin and syringic acid
percentages at different rhizogenetic potential levels.
3. RESULTS
3.1. Total Polyphenols
The total mean content of polyphenols in merithallus
as function of sampling date is presented in Table 1. The
total polyphenol content is expressed as mg of gallic
acid equivalent per g of dry weight (mg GAE·g–1 dw).
Total content of phenolic compounds in Carignan meri-
thallus was the high est in November, but sho wed almost
a constant variation for the rest of sampling period (Ta-
ble 1). By considering the merithallus position on Carig-
nan shoots (apical, central and basal), results showed a
great variation in total polyphenol content (Table 2).
This variation was accentuated in November and char-
acterized by a greatest concentration of polyphenols in
the apical position , which was three-fold high er than that
observed in central and in basal positions.
Table 1. Va riation of total phenolic compounds (T) in Carignan merithallu s as function of sampling date (mg GAE·g–1 dw).
September October November December January
T (mg EAG·g–1 dw) 3.77 ± 0.76b 3.30 ± 0.64d 7.63 ± 0.17a 1.93 ± 0.94e 3.55 ± 0.26c
Values are the means of triplicates ±SD. Values in the same row with different superscript, a-eare significantly different at P < 0.05.
Table 2. Variation of total phenolic content in Carignan merithallus (mg GAE·g–1 dw) as function of their position on vine shoots.
Month of the year
Position September October November December January
Apical 1.76 ± 0.49d 4.18 ± 0.60c 14.73 ± 1.46a 1.21 ± 0.42e 5.22 ± 0.04b
Central 6.06 ± 0.36a 3. 06 ± 0 .01d 5.64 ± 0.84b 1.32 ± 0.68e 3.27 ± 0. 84c
Basal 3.5 ± 0.01a 2.65 ± 0.01c 2.52 ± 0.53cd 3.27 ± 0.52b 2.17 ± 0.26d
Values are the means of triplicates ±SD. Values in the same row with different superscript, a-eare significantly different at P < 0.05.

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3.2. Variation of Individual Phenolic
Compounds
Figure 1 indicates on chromatographic profile (reten-
tion time) of phenolic compounds found in Carignan
merithallus. Nine phenolic compounds (out of 12) were
identified according to their respective retention time as
follows: 1) gallic acid, 2) caffeic acid, 3) dihydroxy-
phenylacetic acid, 4) chlorogenic acid, 5) syringic acid,
6) ferulic acid, 7) naringin, 8) quercetin, 9) coumarin.
Percent distribution of analyzed phenolic compounds
within shoots of Carignan merithallus as function of
sampling date is presented in Table 3. We noted that the
content of some phenolic compounds (ferulic acid and
coumarin) was proportional to the v ariation of total con-
tent in merithallus. On the other hand, quercetin and
dihydroxyphenylacetic acid contents increased with time
in contrast to chlorogenic acid. Gallic acid percentage in
the central part of shoots increased steadily to reach
highest values in December before decreasing. For the
same position, caffeic acid was highly expressed in No-
vember while the concentrations of naringin and syringic
acid showed a sawtooth-shaped variation (Ta bl e 3). As
the variation of non-id entified compounds present in the
chromatogram was not stable over time, only phenolic
compounds that have significant correlations with the
rhizogenetic potential were co nsidered for fu rther invest-
tigation.
3.3. Correlation between Phenolic
Compounds and Rhizogenetic
Potential
When addressing the variation of % coumarin and
carignan rhizogenetic potential with respect to sampling
date [28], we noticed that there was a strictly negative
correlation for merithallus in basal position (Figure 2).
Our investigation showed a moderately positive correla-
tion between syringic acid and the rhizogenetic potential
(Figure 3). There is also a positive correlation between
naringin and rhizogenetic potential but not significant
(Figure 4).
Figure 1. Chromatographic profile of phenolic compounds found in Carignan shoots. 1. Gallic acid; 2. Caffeic acid; 3. Dihydroxy-
phenylacetic acid; 4. Chlorogenic acid; 5. Syringic acid; 6. Feulic acid; 7. Naringin; 8. Coumarin; NI. Non identified.
(a) (b)
Figure 2. Evolution of the coumarin in the merithallus (a) and correlation between these percentages in basal position and the
rhizogenetic potential (b).

K. D. Zohra et al. / Agricultural Sciences 2 (2011) 497-504
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(a) (b)
Figure 3. Evolution of syringic acid in the merithallus (a) and correlation between these percentages and the rhizogenetic potential (b).
(a) (b)
Figure 4. Evolution of the naringin of the merithallus (a) and correlation between these percentages and the potential rhizogenetic
according to the date of taking away (b).
4. DISCUSSION
Interactions between phenolic compounds, organo-
genesis and plant growth have been studied frequently as
mentioned in the introduction section. The regulation of
the endogenous rate of auxin as part of the enzymatic
activity of auxin-oxydase, and auxin transport are two
important events.
The polarized transport of auxin has been studied by
several authors [29-32]. However, this transport can be
disturbed or controlled by some phenolic compounds. In
terms of regulation of polarized transport, the role of
some specific phenolic compounds (phytotropins) is the
most evident as they compete with efflux transporters of
auxin according [29].
The variation of percent coumarin in Carignan and the
rhizogenetic potential as function of sampling dates [22 ]
showed a significant negative correlation for the basal
position of the merithallus on vine shoots. Syringic acid
and naringin can also co rrelate positively with the rhizo-
genetic potential. We noticed also that the quercetin
content increased during the phase of dormancy of buds
and reached highest levels by January (Ta bl e 3). Darne
and Atalay [33] reported that there was a direct correla-
tion between the rhizogenetic capacity and the synthesis
of phenolic compounds in vine shoots.
By studying the adventive rhizogenesis in Pinus con-
torta, Brinker et al. [32] noticed that the active transport
of auxin is reduced during the first phase of the rhizo-
genesis. They also observed a reduction in the transcript-
tion of the gene which codes for the kinase-like protein,
involved in the control of auxin transport. Intermediate
products resulting from the biosynthesis of phenolic
compounds may accumulate and create a barrier that
inhibits auxin transport. Brinker et al. [32] indicated that
treatments with auxin activate the cellular division. This
hormone has a major role in the initiation and the de-
velopment of adventive roots. The mechanism utilizes
phenolic compounds as a barrier that prevents auxin
transport. This natural barrier consists of a protein of

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alue
Table 3. Percent distribution (%, w/w) of analyzed phenolic compounds within shoots of Carignan merithallus as function of sampling date.
Vs are the means of triplicates ±SD. Values in the same row with different superscript, a-kare significantly different at P < 0.05. Co mp ou nd s : 1: Gallic acid, 2: cafeic ac i d, 3: NI, 4: dihy droxypheny l ac et i c acid, 5:
N
I, 6: chlorogenic acid, 7: NI, 8: syringic acid, 9: ferrulic acid, 10: Naringin, 11: Coumarin.

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having a flavonolic origin [30,32] noticed that the tran-
scription of the gene coding for this enzyme is affected
during the pha se of radial primordial formation.
5. CONCLUSIONS
The evolution of phenolic compounds was studied on
Carignan merithallus during the phases of bud dormancy
and end of dormancy. Our outcomes indicate that rhizo-
genesis coincides with quercetin synthesis. This phenolic
compound belongs to the class of phytotropins according
to Brunn et al. [29]. We observed also a negative corre-
lation between coumarin and rhizogenetic potential of
Carignan vine. In contrast, positive correlations were
found with naringin and syringic acid.
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