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Acta Botanica Gallica
ISSN: 1253-8078 (Print) 2166-3408 (Online) Journal homepage: https://www.tandfonline.com/loi/tabg20
Micropropagation of carob, Ceratonia siliqua L., by
apex culture
Micropropagation du caroube, Ceratonia siliqua L., par cultures de
méristèmes
Souheila Naghmouchi , Mohamed Laarbi Khoudja , Agusti Romero &
Mohamed Boussaid
To cite this article: Souheila Naghmouchi , Mohamed Laarbi Khoudja , Agusti Romero &
Mohamed Boussaid (2012) Micropropagation of carob, Ceratonia�siliqua L., by apex culture, Acta
Botanica Gallica, 159:3, 357-361, DOI: 10.1080/12538078.2012.737124
To link to this article: https://doi.org/10.1080/12538078.2012.737124
Published online: 26 Nov 2012.
Submit your article to this journal
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Micropropagation of carob, Ceratonia siliqua L., by apex culture
Micropropagation du caroube, Ceratonia siliqua L., par cultures de méristèmes
Souheila Naghmouchi
a,b
*, Mohamed Laarbi Khoudja
a
, Agusti Romero
c
and Mohamed Boussaid
b
a
Institute of Research in Rural Engineering, Water and Forestry (INRGREF), rue Hedi Karay, Ariana BP10, 2080 Tunis, Tunisia;
b
National Institute of Sciences Applied to Technology (INSAT Tunis), Centre Urbain Nord, BP 676, 1080 Tunis, Tunisia;
c
Institute of
Research in Agroalimentary Technology (IRTA), Centre de Mas bové BP 415, 43280 Reus, Spain
Abstract: The great variability of Ceratonia siliqua, its sexual polymorphism and its long stage of juvenility imply the use
of conventional and in vitro culture techniques. The high phenotypic variation between carob cultivars has important
implications for selection, cultivation practices and establishment of new plantations to improve productivity of this crop.
The present work was undertaken to develop a simple protocol for micropropagation of carob through meristem culture on
half-strength Murashige and Skoog medium. The best results were obtained by introducing the meristem culture when
buds were starting to swell. Meristem sprouting was stimulated on Murashige and Skoog medium supplemented with
2,4-dichlorophenoxyacetic acid. The highest number of well-developed shoots from meristems was obtained with 2 mg l
–1
6-benzylaminopurine and 2 mg l
–1
2,4-dichlorophenoxyacetic acid. Higher rooting ability and root quality were obtained
with 4 mg l
–1
naphthalene acetic acid.
Keywords: Ceratonia siliqua; meristem; micropropagation; plant growth regulators; rooting
Résumé: La forte variabilité génétique, le polymorphisme sexuel et la longue phase juvénile du caroubier (Ceratonia
siliqua) nécessitent d’avoir recours à la multiplication végétative par des techniques conventionnelles ou par culture in
vitro. La forte variabilité entre les cultivars de caroubier a d’importantes implications pour la sélection et pour les
pratiques de culture et d’établissement de nouvelles plantations en vue d’améliorer la productivité de cette plante. Le
présent travail a été entrepris pour développer un protocole simple de micropropagation du caroubier par culture de
méristèmes sur milieu de Murashige et Skoog dilué de moitié. Les meilleurs résultats ont été obtenus avec des
méristèmes prélevés sur des bourgeons au début de leur débourrement. Le débourrement des méristèmes a été stimulé
sur le milieu de Murashige et Skoog en présence d’acide 2,4-dichlorophénoxyacétique. Le meilleur résultat en termes de
nombre de pousses bien développées a été obtenu avec 2 mg de l–1 6-benzylaminopurine et 2 mg de l–1 acide 2,4-
dichlorophénoxyacétique Le pourcentage d’enracinement le plus élevé a été obtenu en présence de 4 mg de l–1d’acide
naphthalène acétique.
Mots clés: Ceratonia siliqua; enracinement; méristème; micropropagation; régulateurs de croissance
Introduction
The carob tree (Ceratonia siliqua L.), belonging to the
family Fabaceae, is widely used in Mediterranean
countries (Rejeb 1989; Tous and Battle 1990) and is
cultivated for ornamental and industrial purposes. It is
mainly used in the pharmaceutical and liquor industries
(Haselberg 2000; Naghmouchi et al. 2009).
Carob is a good candidate for in vitro propagation
because aging tree cuttings are difficult to root (Alorda
and Medrano 1988; Naghmouchi 2011). A
micropropagation system via cuttings has been
successfully developed for carob tree (Romano, Barros,
and Martins-Loucao 2002; Custódio, Carneiro, and
Romano 2004; Naghmouchi et al. 2008).
In vitro contamination represents a serious problem
with carob. Trees in the field have been growing in open
conditions for extended periods of time and their organs
are frequently contaminated both externally and internally
by various microorganisms. Most of the organisms that are
encountered are of no particular importance to the plant
in vivo but result in contamination when cultured in vitro
because bacteria and fungal spores will grow rapidly on a
rich culture medium (Cassells 1991). Consequently, the first
step in preparing a plant for tissue culture is to eliminate
*Corresponding author. Email: den_souheila@yahoo.fr
Acta Botanica Gallica: Botany Letters
Vol. 159, No. 3, September 2012, 357–361
Société botanique de France
ISSN 1253-8078 print/ISSN 2166-3408 online
Copyright Ó2012 Société botanique de France
http://dx.doi.org/10.1080/12538078.2012.737124
http://www.tandfonline.com
microorganisms. Meristem culture is often useful for this
purpose (Cassells 1991). Depending on meristem size,
viruses can also be eliminated. Hence, meristems are often
used as initial explants for large-scale micropropagation.
The problem of contamination in carob plant tissue culture
hasbeenreviewedbyNaghmouchi(2011).
Adventitious root formation is also essential for
successful vegetative propagation of many woody plants.
However, for several tree species, rooting remains a
major problem. Some progress has been made using
different chemical or natural components in the rooting
media such as auxins combined with phenolic
compounds (Onay et al. 2003; Fotso et al. 2007).
The literature on in vitro culture of carob through
meristem tips is sparse. Therefore the aim of this paper
was to gain information on meristem tip culture of carob
as well as to determine the optimal balance of plant
growth regulators to ensure its continual growth.
Materials and methods
Plant sources and cultivation
Plant material
Plant material used in these experiments was obtained
from dormant shoots collected from 12-year-old carob
trees, growing in the carob collection of the Botanical
Garden of Research Institute in Rural Engineering, Water
and Forestry (INRGREF, Tunisia).
Disinfection and bud dissection
Apical shoots (5–10 cm long) were transferred to the
laboratory; the larger leaves were removed, the surface
was washed carefully with water and Tween-20. The
explants were shaken in 70% ethanol for 5 min and then in
a 20% sodium hypochlorite solution for 20 min. Finally,
stems were rinsed three times with sterile water.
The isolation of meristem tip was carried out under
aseptic conditions. With a binocular loupe, the meristem
tip (0.3–0.7 mm long), composed of the apical dome and
one or two leaf primordia, was dissected and explanted.
Media and culture conditions
Murashige and Skoog (1962) macronutrients and
micronutrients half-strength (MS/2) were used for all
experiments, supplemented with 3% saccharose and
solidified with agar (0.8%). Myoinositol, thiamine and
nicotinic acid were added to the medium and the pH
was adjusted to 5.7 after adding plant growth regulators.
Experimental design and statistical analyses
To determine if bud dormancy affects meristem
establishment, meristem tips of carob were introduced at
different dates (from September to May).
To stimulate the meristem sprouting, 6-
benzylaminopurine (BA) and 2,4-dichlorophenoxyacetic
acid (2,4-D) were combined in 25 different treatments.
Explants were cultured for 16 h at 23°C under
illumination with white fluorescent light (55 μmol m
–2
s
–1
)
followed by 8 h at 21°C in the dark.
Surviving explants were subcultured every 2 weeks.
After 8 weeks, the sprouting rate of meristems was
recorded.
When the developed shoots reached 5 mm in
diameter, they were dissected and transferred to fresh
media to start the proliferation and elongation stage.
Fifteen different combined treatments of BA and 2,4-D
were tested; the number and length of shoots were
recorded after a 5-week culture as described before for
each treatment. For the rooting stage, indol-3-butyric
acid (IBA), indol-3-acetic acid (IAA) and naphthalene
acetic acid (NAA) were tested at 1, 2 and 4 mg l
–1
, the
rooting rate and the number and length of roots were
established after 6 weeks under the same culture
conditions.
An analysis of variance was conducted to estimate
the influence of introduction date on sprouting of
meristems, the effects of plant growth regulators
concentrations on sprouting, length and number of
shoots in the proliferation stage, as well as rooting,
number and length of roots in the rooting stage. There
were four replications for each experiment.
Results
Meristem sprouting
The date of carob meristem introduction significantly
influenced (p< 0.001) the survival and length of
shoots obtained after 8 weeks of culture. Survival of
explants was higher (96%) between February and May
and the shoots developed were much longer (1.09 cm).
On the other hand, the survival of explants (20%)
cultured between September and January as well as
the length of microshoots developed (0.12 cm) were
very low.
All tested 2,4-D concentrations stimulated a fast
sprouting of meristem tips (Table 1). The MS media
supplemented with 2,4-D at 1, 2 and 4 mg l
–1
gave the
highest rates of meristem sprouting (100, 94 and 96%,
respectively).
The MS medium supplemented with 2 mg l
–1
BA
stimulated bud formation, but, combined with 2,4-D,
sprouting rate decreased with higher BA concentrations.
The BA and 2,4-D concentrations significantly
influenced (p< 0.05) the development of leaves from
meristem tips of C. siliqua. The absence of 2,4-D in the
media prevented meristems from developing leaves, and
these explants died within the first 6 weeks.
Shoot proliferation
Explant shoot number after 6-week transplantation
responded to concentration of BA (Table 2). The
maximum number of shoots was obtained with 2 mg l
–1
BA supplemented with different concentrations of 2,4-D.
358 S. Naghmouchi et al.
Shoot length after 6 weeks in culture increased
significantly with 2,4-D (p< 0.05) (Table 2). The longest
explants were found in the MS medium supplemented
with 2 mg l
–1
2,4-D and the shortest in medium
supplemented with 0.5 mg l
–1
2,4-D, whatever the
concentrations of BA.
Rooting of microshoots
Microshoots excised from proliferating cultures were
cultured on MS/2 medium devoid of growth regulators
or with auxin. Microshoots started to form roots within
3–4 weeks. The percentage of rooted microshoots, and
the number and the length of roots per culture were
influenced by the different types and concentrations of
auxin. When no auxin was added to the medium, no
roots were formed (Table 3).
Microshoots explanted with IBA and NAA produced
normal roots and formed very little callus on the base of
the explant after 1 month culture. With IAA, roots were
abnormal, thick, and some grew out onto the surface of
the agar. Moreover, rooting with this form of auxin gave
rise to an excessive amount of callus formed at the base
of the shoots and the rooting ability was very modest
(3.33% with 2 mg l
–1
IAA).
Among the three auxins, NAA at 4 mg l
–1
produced
the highest percentage of rooting (53.33%), the highest
number of roots per shoot (10.68) and the maximum
average root length (3.04 cm).
Discussion
The most widespread method to establish plant material
in vitro is by use of stem nodal segments (Romano,
Barros, and Martins-Loucao 2002; Gonçalves et al.
2005; Naghmouchi et al. 2008; Brugaletta et al. 2009).
In our experiments with carob, axillary shoot culture was
avoided because of high contamination.
Establishing carob plantlets in vitro through
meristem tip culture is a time-consuming technique;
however, the contamination rates decreased drastically
(less than 5% of contaminated explants) and no
internal contaminant was observed after the cultures
were established.
Few indications for the best time for meristem
carob introduction have been quoted in the literature
(Lengliz et al. 2007). Boxus and Quoirin (1974) found
that the percentage of Prunus species sprouting
depends on bud dormancy. Similar results were
obtained by Carré et al. (1979) working with
strawberries, although the optimum season for
introduction was different. Our results show that the
best time for carob meristem introduction is the period
from February to May, coinciding with the vegetative
growth of carob and bud swelling. So introduction
should not be performed during bud dormancy (from
September to January).
As previously reported by Saidi, Lamarti, and
Badoc (2007), our results show that 2,4-D is needed in
Table 1. Effect of BA and 2.4-D concentrations (mg l
–1
) on meristem sprouting after four subcultures of 2 weeks on MS/2.
Tableau 1. Effet des concentrations de BAP et 2,4-D (mg l
–1
) sur le débourrement du méristème après quatre subcultures de 2 semaines sur MS/2.
BA 0 0.5 1 2 4
2.4-D 0 0.5 1 2 4 0 0.5 1 2 4 0 0.5 1 2 4 0 0.5 1 2 4 0 0.5 1 2 4
Sprouting (%) 24 48 100 94 96 14 52 86 74 82 26 14 58 50 36 90 50 26 80 48 42 34 20 38 44
hijk efg a ab a jk efg abc bcd abc hijk k def efg Ghij ab efg hijk abc efg fgh ghijk iijk fghi fgh
BA, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; MS/2, half-strength Murashige and Skoog medium.
Data represent means compared with Duncan’s multiple-range test (p<0.05). Data followed by the same letter in the same column are not significantly different.
Acta Botanica Gallica: Botany Letters 359
the culture medium to promote the development of a
rosette of leaves. In the absence of 2,4-D, meristems
fail to develop shoots and die within the first 6 weeks
of culture.
As BA improves survival of meristems at 2 mg l
–1
,a
correct balance has to be established between BA and
2,4-D for meristem establishment. Indeed, combinations
between BAP (0.5 mg l
–1
) and 2,4-D (1, 2 and 4 mg l
–1
)
give important rates of sprouting.
Shoot elongation and multiplication are facilitated
with 2 mg l
–1
BA and 2,4-D, respectively.
Both IAA and IBA gave lower rooting rates than
NAA and produced excessive callus formation at the
cutting base. These results do not agree with the
previous findings of Romano, Barros, and Martins-
Loucao (2002), Naghmouchi et al. (2008) and Saidi,
Lamarti, and Badoc (2007), who reported that the
preferred auxin for carob root initiation in axillary bud
cultures was IBA.
Conclusion
This study reports successful micropropagation of carob
(Ceratonia siliqua) through apex culture. The system
consisted of three stages: meristem sprouting, shoot
proliferation and elongation, and rooting. Maximum
Table 2. Effect of different BA and 2.4-D concentrations on in vitro shoot multiplication and elongation obtained with carob
meristem tips after 5 weeks culture on MS/2.
Tableau 2. Effet des différentes concentrations de BAP et 2,4-D sur la multiplication et l’élongation in vitro des feuilles obtenues
sur des méristèmes de caroubier après 5 semaines de culture sur MS/2.
Growth regulators (mg l
–1
) Number of shoots/explant Shoot length (cm)
2.4-D BA
0.5 0 1.10 f 0.23 cd
0.5 0.5 1.50 de 0.19 d
0.5 1 2.18 c 0.26 cd
0.5 2 4.98 a 0.28 cd
0.5 4 3.10 b 0.29 c
1 0 1.26 ef 0.40 b
1 0.5 1.68 d 0.42 b
1 1 2.32 c 0.41 b
1 2 5.02 a 0.41 b
1 4 3.04 b 0.39 b
2 0 1.18 ef 0.85 a
2 0.5 1.80 d 0.76 a
2 1 2.22 c 0.81 a
2 2 5.08 a 0.77 a
2 4 3.24 b 0.81 a
BA, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; MS/2, half-strength Murashige and Skoog medium.
Data represent means compared with Duncan’s multiple-range test (p< 0.05). Data followed by the same letter in the same column are not significantly
different.
Table 3. Effects of auxin type and concentrations on rooting of in-vitro-proliferated microshoots of carob after 6-week culture on
MS/2.
Tableau 3. Effet du type et de la concentration de l’auxine sur l’enracinement des micropousses de caroubier proliférées in vitro
après 6 semaines de culture sur MS/2.
Auxin (mg l
–1
) Rooting (%)
Root number/
explant Root length (cm) Callus formation
⁄
Test 0.00 d 0.00 F 0.00 d ++
IBA 1 6.66 d 1.67 Ef 0.50 bc –
2 10.00 cd 1.40 Ef 0.38 c +
4 26.67 b 5.47 C 2.15 ab –
NAA 1 3.33 d 2.50 De 0.45 b –
2 23.33 b 8.31 B 2.81 a +
4 53.33 a 10.68 A 3.04 a –
IAA 1 0.00 d 0.00 F 0.00 d –
2 3.33 d 2.00 def 0.90 b +++
4 0.00 d 0.00 F 0.00 d –
IAA, indol-3-acetic acid; IBA, indol-3-butyrinc acid; MS/2, half-strength Murashige and Skoog medium; NAA, naphthalene acetic acid.
⁄
Intensity of callus formation on the base of explant: –, no; +, many; ++, moderate; +++, high callus formation.
Data were compared with Duncan’s multiple-range test (p< 0.05). Data followed by the same letter in the same column are not significantly different.
360 S. Naghmouchi et al.
sprouting is achieved on MS/2 medium containing 2,4-
D. Shoot proliferation and elongation occurs after
transferring shoots to the same medium containing
2mgl
–1
2,4-D and 2 mg l
–1
BA. Rooting of shoots is
obtained on MS/2 medium supplemented with 4 mg l
–1
NAA.
This regeneration system can be used to produce
carob plants within a short time period.
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Acta Botanica Gallica: Botany Letters 361
