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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.
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Acta Botanica Gallica
ISSN: 1253-8078 (Print) 2166-3408 (Online) Journal homepage:
Micropropagation of carob, Ceratonia siliqua L., by
apex culture
Micropropagation du caroube, Ceratonia siliqua L., par cultures de
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
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Published online: 26 Nov 2012.
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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
*, Mohamed Laarbi Khoudja
, Agusti Romero
and Mohamed Boussaid
Institute of Research in Rural Engineering, Water and Forestry (INRGREF), rue Hedi Karay, Ariana BP10, 2080 Tunis, Tunisia;
National Institute of Sciences Applied to Technology (INSAT Tunis), Centre Urbain Nord, BP 676, 1080 Tunis, Tunisia;
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
6-benzylaminopurine and 2 mg l
2,4-dichlorophenoxyacetic acid. Higher rooting ability and root quality were obtained
with 4 mg l
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 davoir recours à la multiplication végétative par des techniques conventionnelles ou par culture in
vitro. La forte variabilité entre les cultivars de caroubier a dimportantes implications pour la sélection et pour les
pratiques de culture et détablissement de nouvelles plantations en vue daméliorer 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 dacide 2,4-dichlorophénoxyacétique. Le meilleur résultat en termes de
nombre de pousses bien développées a été obtenu avec 2 mg de l1 6-benzylaminopurine et 2 mg de l1 acide 2,4-
dichlorophénoxyacétique Le pourcentage denracinement le plus élevé a été obtenu en présence de 4 mg de l1dacide
naphthalène acétique.
Mots clés: Ceratonia siliqua; enracinement; méristème; micropropagation; régulateurs de croissance
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 difcult 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 eld 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 rst
step in preparing a plant for tissue culture is to eliminate
*Corresponding author. Email:
Acta Botanica Gallica: Botany Letters
Vol. 159, No. 3, September 2012, 357361
Société botanique de France
ISSN 1253-8078 print/ISSN 2166-3408 online
Copyright Ó2012 Société botanique de France
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
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 (510 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 (0.30.7 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
solidied 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 uorescent light (55 μmol m
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
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
, the
rooting rate and the number and length of roots were
established after 6 weeks under the same culture
An analysis of variance was conducted to estimate
the inuence 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.
Meristem sprouting
The date of carob meristem introduction signicantly
inuenced (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
gave the
highest rates of meristem sprouting (100, 94 and 96%,
The MS medium supplemented with 2 mg l
stimulated bud formation, but, combined with 2,4-D,
sprouting rate decreased with higher BA concentrations.
The BA and 2,4-D concentrations signicantly
inuenced (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 rst 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
BA supplemented with different concentrations of 2,4-D.
358 S. Naghmouchi et al.
Shoot length after 6 weeks in culture increased
signicantly with 2,4-D (p< 0.05) (Table 2). The longest
explants were found in the MS medium supplemented
with 2 mg l
2,4-D and the shortest in medium
supplemented with 0.5 mg l
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
34 weeks. The percentage of rooted microshoots, and
the number and the length of roots per culture were
inuenced 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
Among the three auxins, NAA at 4 mg l
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).
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
) 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
) 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 Duncans multiple-range test (p<0.05). Data followed by the same letter in the same column are not signicantly 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 rst 6 weeks
of culture.
As BA improves survival of meristems at 2 mg l
correct balance has to be established between BA and
2,4-D for meristem establishment. Indeed, combinations
between BAP (0.5 mg l
) and 2,4-D (1, 2 and 4 mg l
give important rates of sprouting.
Shoot elongation and multiplication are facilitated
with 2 mg l
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 ndings 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.
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 lélongation in vitro des feuilles obtenues
sur des méristèmes de caroubier après 5 semaines de culture sur MS/2.
Growth regulators (mg l
) 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 Duncans multiple-range test (p< 0.05). Data followed by the same letter in the same column are not signicantly
Table 3. Effects of auxin type and concentrations on rooting of in-vitro-proliferated microshoots of carob after 6-week culture on
Tableau 3. Effet du type et de la concentration de lauxine sur lenracinement des micropousses de caroubier proliférées in vitro
après 6 semaines de culture sur MS/2.
Auxin (mg l
) 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 Duncans multiple-range test (p< 0.05). Data followed by the same letter in the same column are not signicantly 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
2,4-D and 2 mg l
BA. Rooting of shoots is
obtained on MS/2 medium supplemented with 4 mg l
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
... Micropropagation of carob by axillary budding from nodal explants and apex culture was reported also by Custódio et al. [10] and Gonçalves et al. [11]. However, Naghmouchi et al. [12,13] regenerated multiple shoots from the apical and nodal shoots, but with a limited growth. According to Romano et al. [4], rooting and acclimatization was observed by modifying the medium composition, but on certain carob genotypes. ...
... The results also agree with the previous findings of Romano et al. [4] and Naghmouchi et al. [12], who reported that the preferred auxin for carob root initiation in axillary bud cultures was IBA. In addition, Ricci et al. (2016) and Shahzad et al. [5] used IBA to induce rooting in carob, while, Naghmouchi et al. [13] showed root induction using all the three auxin treatments (IBA, NAA and IAA). Trp was added to the root induction medium to induce the endogenous level of auxin. ...
Full-text available
Carob (Ceratonia siliqua, Fabaceae) is a Mediterranean tree with socioeconomic and ecological interests. The conventional propagation of carob is difficult, therefore, micropropagation offers an efficient alternative to respond to the increasing demand for this valuable plant. In vitro culture establishment of explants from an adult tree was successfully performed on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 2.46 μM 2-isopentenyladenine (2iP) for stem node segments, while direct organogenesis from leaf segments was optimum on MS medium supplemented with 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ). Leaf segments produced twice the number of shoots (10.67 shoots/explant), compared to the stem node segments. The highest proliferation of shoots was observed on MS medium supplemented with 8.9 μM BAP and 2.46 μM 2iP. Root induction was observed on the shoots cultured on MS medium supplemented with 9.8 μM indole-3-butyric acid (IBA) and 1.9586 μM tryptophan (Trp) on 86.67% of shoots. Well rooted plantlets were successfully acclimatized to the greenhouse. This report describes an improving protocol for the micropropagation of carob to respond to the needed efforts to cultivate carob in Egypt.
... Different explant types such as stem nodal segments [5,16,18,38,47], meristem tip culture [39], hormone concentrations [18,35,38,39,45,47,49,52,53,62] and basal media [1,5,16,37,60] have statistically important effect on regeneration and callus formation. ...
... Different explant types such as stem nodal segments [5,16,18,38,47], meristem tip culture [39], hormone concentrations [18,35,38,39,45,47,49,52,53,62] and basal media [1,5,16,37,60] have statistically important effect on regeneration and callus formation. ...
Full-text available
Ceratonia siliqua L. is a slow growing evergreen tree of the family Fabaceae used for the rehabilitation of marginal and submarginal dry areas of the Mediterranean basin due to it's resistant to drought and salt tolerance. In this study, the effects of different basal media (Woody Plant Medium and Murashige and Skoog medium), explant types (cotyledon and hypocotyl) and growth regulators (BA, Kinetin and NAA) on in vitro callus formation, differentiation of callus to shoot and root formation were investigated. High frequencies of caullogenesis were obtained and the best medium for callus induction was WPM supplemented with 1.0 mg L-1 BA + 0.5 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA and 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA for hypocotyl explants and 0.5 mg L⁻¹ BA + 1.0 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA; 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA and 1 mg L⁻¹ Kinetin + 0.5 mg L⁻¹ NAA for cotyledon explants. Callus induction was more readily obtained from hypocotyl explants than cotyledon explants. It was determined that explant types as significant on shoot formation, statistically. The shoot ratio was obtained from cotyledon explants in WPM as 10%. The best regeneration was obtained from cotyledon explants placed on WPM (30%) instead of MS medium.
... Culture of different carob explants: from parts of young seedlings (cotyledonary leaves, hypocotyl, roots, meristems, lateral and axillary buds) [12][13][14][15][16][17][18][19][20][21], meristems, lateral and axillary buds of adult female trees [4,13,[22][23][24][25][26][27], ova taken from female flowers [28], anthers of the male inflorescences of seed trees [29], immature seeds [30], mature cotyledons of seeds [31] and fruit tissue [32] is often accompanied by the formation of a callus whose importance varies according to the conditions studied. ...
... These results are in concordance with those obtained in the study of callogenesis from apex of young seedlings. The callogenous capacity of explants from mature trees was also ed by other researchers [4,13,[23][24][25][26][27]. ...
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Callus induction was successfully carried out from several explants of carob (Ceratonia siliqua L.). Callogenesis from the apex was tested on three different media containing Woody Plant Medium (WPM), Murashige and Skoog (MS) or Schenk and Hildebrandt (SH) macronutrients supplemented with two different hormonal solutions: benzylaminopurine (BAP) at 4.44 μM alone, or 2.22 μM of BAP plus 5 μM of 2-naphthalineacetic acid (NAA). Primary callus formation was obtained on a medium containing 88% WPM macronutrients. Callus formation from other parts of the plant was as follows: Cotyledon embryos extracted from immature seeds (85% success rate on WPM medium, containing 4.44 μM BAP and 5 μM NAA); Cotyledon leaves taken from 7-day-old seedlings, obtained from in vitro germination of seeds (62% success rate on WPM medium, containing 4.44 μM BAP and 5 μM NAA).
... [28] and using micropropagation by shoot tip cultures [29]. Other studies proved also that high concentrations of auxins combined with BAP and GA 3 give good results in the culture of young buds [26], as well as for apical buds break from a mature tree [21]. Radi [32], this step is optional and is only required for very small shoots in order to obtain elongated leafy stems of Pyros malus which are then used for rooting. ...
... The inhibitory effect of high concentration of BAP was observed in other studies:Belaizi et al. (1995) [20] noticed that during the study of carob regeneration from lateral buds, concentrations higher than 4.44 µM does not increase the number of broken buds. As well, Romano et al.(2002) [6] affirm that the best multiple shoot response was obtained in MS medium supplemented with 4.44 µM BAP, for in vitro propagation protocol based on axillary bud proliferation.Also, survival of meristems reaches the maximum with 8.88 µM BAP and decreases in a higher concentration[21]. However,Radi et al. (2013) [22] found that MS medium added with 8.88 µM BAP affects significantly the size of shoots obtained from lateral buds and gives the best results among lower concentrations. ...
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Adventitious budding from embryonic cotyledons of immature seeds of carob was obtained. The combination of 4.44 µM 6-benzylaminopurine (BAP) and 1.5 µM 1-Naphthaleneacetic acid (NAA) furthers the neoformation of adventitious buds. These latter multiply on Murashige and Skoog medium (MS) added with 2.22 µM BAP, stems and leaves growing was improved by adding 2.02 µM of Gibberellic acid (GA3). Elongation was favored by NAA at 0.5 µM. 70 % of rooting was obtained with 10 µM of Indole-3-butyric acid (IBA).
... Micropropagation is currently considered as one of the most effective techniques for the massive multiplication of plants. In vitro propagation of carob, despite being studied by different researchers (El Shafey et al., 1998;Vinterhalter and Vinterhalter, 2003;Vinterhalter et al., 2007;Saidi et al., 2007;Brugaletta et al., 2009;Hsina and El-Mtili, 2009;Hakim et al., 2010;Naghmouchia et al., 2012), still shows some difficulties that do not allow the inclusion of these technique on a large scale. Among these, the recalcitrance of adult material to in vitro multiplication, the bacterial contamination of endogenous origin, browning, the slow rate of proliferation and the low rate of acclimatization in vivo, in part affect the success of the culture and require further research efforts. ...
... In fact, when nodes with dormant bud from woody larger branches, or to a lesser extend, apical shoots of young branches are used to begin in vitro culture, a consistent loss of explants during in vitro culture is observed in relation mainly to the browning phenomenon (55-60% of the explants). Moreover, adult plant organs are frequently contaminated both externally and internally by various microorganisms which are not particularly important in vivo but result in high contamination when cultured in vitro (Naghmouchia et al., 2012). This is not true for seedling apices since a low rate of contamination was observed (<10%) even after the simple sterilization procedure previously described. ...
... Thus, single explant harvested from mature tissues could generate only one shoot that too with a very limited growth. However, Naghmouchi et al. (2008Naghmouchi et al. ( , 2012 got some success in multiple shoot regeneration through the apical shoot and nodal culture but with a limited success in growth and development of the regenerants. According to Mohamed-Yasseen et al. (1992) shoot tip taken from aseptically germinated seedlings produced 5 shoots per explant. ...
... The use of IBA has been reported to induce rhizogenesis in many plant species (George 1993) and is described to be more resilient to catabolism (Epstein and Ludwig-Muller 1993) which could be the reason for its better performance in inducing roots as compared to IAA and NAA. While, in carob, Naghmouchi et al. (2012) reported root induction in all the three auxin treatments. However, transfer to basal medium after IBA treatment (Romano et al. 2002) and a combination of IBA + 5-BDPU (Ricci et al. 2016) was found to induce rooting in microshoots. ...
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Key message The article describes refined regeneration process in Ceratonia siliqua using different plant growth regulators along with antioxidant, SEM and IRGA analysis to understand the developmental behaviour of the plantlets. Abstract The present study describes a simplified seed germination process in Ceratonia siliqua L. and gives a comparative analysis of hormonal supply for enhanced adventitious shoot regeneration through aseptic seedling-derived cotyledonary nodes. The axillary bud induction and multiplication was greatly influenced by the concentration and type of cytokinin viz. 6-benzylaminopurine (BA), meta-Topolin (mT), Kinetin (Kn) and Thiadiazuron (TDZ). Adenine-based cytokinins, BA and mT, augmented in Murashige and Skoog medium, were proved to be more responsive. The medium containing 10 µM BA produced a maximum of 9.1 ± 3.0 shoots/ explant. A combination of optimal concentration of BA with NAA (0.5 µM) however, resulted into a significant production of 28.10 ± 0.14 shoots per CN. Half-strength MS + IBA (10 µM) in the presence of light ensured more number of root formation while half-strength MS + IBA (5.0 µM) and stimulation in the dark for 1 week ensured better root growth (4.98 ± 0.08 cm). Plantlets were successfully acclimatized in soilrite™, showing 65% survival. The work is supported with the studies based on antioxidant enzyme activity as well as on net photosynthetic rate and its related attributes in comprehension with SEM analysis of leaves showing stomatal micromorphology during three stages: in vitro, during acclimatization and in net house conditions. The studies during acclimatization reflect better understanding of the stabilization of micropropagated plantlets to the environmental conditions.
... Indeed, since the 1990s, different types of explants have been used from either young seedlings or adult tree. Also, apex culture from young seedlings has been studied [21] [22] [23] [24]. This technique allows working on explants without prior disinfection and offers several possibilities of multiplication. ...
This datasheet on Ceratonia siliqua covers Identity, Overview, Associated Diseases, Pests or Pathogens, Distribution, Dispersal, Biology & Ecology, Environmental Requirements, Uses, Management, Genetics and Breeding, Food Quality, Economics, Further Information.
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Micropropagation of Ceratonia siliqua (carob) from the apex of 7 days old seedlings was attempted on different growth media based on WPM, Durzan 'modified or not', SH, B5, MS, MS/2, modified Heller, BTM and GD supplemented with microelements, vitamins and MS. Four differents auxins: AIA, AIB, ANA and 2,4D were tested in conjunction with BAP in WPM for the stimulation of bud development. BAP at 0.5 mg/l as well as cytokine favored bud development mainly in WPM and MS media. Similarly combination of BAP at 0.5 mg/l and AIB or AIA at 0.1 mg/l has given good results. Multiplication was stimulated at low concentrations of AIB (0.1 mg/l) and direct rooting of obtained plantlets was better without any auxin pretreatment in MS/2.
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In this study, the success of in vivo and in vitro micrografts of pistachio (Pistacia vera L. cv. "Siirt") materials are presented. The only variable tested was age (1, 5, 10, and 30-year-old trees). Ten- to 12-day-old axenic seedlings germinated in vitro or seedlings (3 to 5 months-old) grown in pots in vivo were used as rootstocks. Shoot tips collected from the four age classes of mature trees of pistachio were the source of scions. Firm contact between the scion and rootstock was assured through the use of parafilm tape at the graft junction for in vivo micrografts. The in vivo micrografting system provided good growth and development for new axillary shoots. These plantlets were successfully transplanted and no problems were encountered with the establishment of micrografted plants in soil. The recovery of microscions was slow, but the use of micrografts onto herbaceous rootstocks proved a useful technique.
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The carob tree (Ceratonia siliqua L.) is an ancient element of the Mediterranean cultural vegetation. Flowering in the evergreen leguminous tree is polygamous dioecious. After bloom in autumn, fruits develop during 10 to 11 months and drop at the end of summer. The brown pods contain 5 to 12 seeds surrounded by a sugary pulp. Important characteristics of the tree are an expressed drought and heat tolerance and a sensitivity towards frost. Formerly, carob pods were applied for animal feeding and human nutrition, whereas nowadays, the crop value is mainly determined by the seed endosperm, which is processed to a stabilizer and thickener (carob bean gum) for the food industry. Portugal is among the leading carob producing countries with the production being localized mainly in the southern province Algarve. Here, carob is traditionally grown under rainfed, often marginal conditions. Objectives of this study were to extend the knowledge on flowering and fruiting in the species with foci on the origin and effects of its alternate bearing behaviour and on aspects of pollination and fertilization on microscopic level. Emphasis was also given to the limiting effects of site conditions on growth. The study was mainly carried out during 4 consecutive growth periods with 30 to 40- year-old trees of the principal Portuguese cultivar 'Mulata' at 4 sites in South Portugal: Pegada (Tavira) near the coast and Freixo (Benafim) in the upper Barrocal, Junqueira in the schistous Serra region and Moreanes in the Southern Alentejo. Vegetative growth varied between years and sites. Course and extend of shoot growth was affected by amounts and distribution of annual rainfall, and also by the fruit load of the respective branches. In alternate bearing trees, vegetative growth displayed a pattern corresponding to the biennial alternation. Reproductive growth features, i.e. numbers of inflorescences, the course of flowering, and the crop size showed large variations from year to year at the different sites. Trees differred considerably in their potential for flower and fruit production dependent on the specific site conditions. The Pegada orchard was most produceful, while at Freixo and Junqueira, yields were lower and less stable. At Moreanes, yields were most meagre and irregular. The main factors for site differentiation were extreme temperatures, such as frost or excessively high temperatures at Moreanes, and inappropriate soil conditions, like the slopy and stony soils at Junqueira which increased the effect of drought. Considering a future extention of rainfed carob farming to marginal sites, only locations with favourable microclimate ought to be selected in order to reduce the chilling related production risk. For locations in the Serra, meliorations and terraces are recommended. In hand-pollinated flowers, first ovule penetration took place 48 h after pollination, and fruitlet growth was initiated another 48 h later. Pollen tubes required 5 d to pass the whole pistil. With increasing distance from the stigma, pollen tubes in the pistil showed a strong basipetal gradient in growth velocity and density. This corresponded with the pattern of seed set in the pods, where disposition of developmental failure in ovules that were distant from the stigma was increased. Non-vital pollen grains and irregularities in tube growth occurred frequently. Tube growth was enhanced at moderate air temperature of 21 to 22°C compared to 31 to 33°C. There was evidence for a considerable ovule longevity even at high temperatures. Abscission of flowers and fruits occurred during three main periods. Pronounced premature shedding of flowers and inflorescences often exceeded 50 %, final fruit set was mostly between 2 and 7 %. Pod growth followed a sigmoidal curve. Seed dry matter accumulation was completed early in the season, when only half of the pericarp dry matter was accumulated. The extended abscission period of mature pods, inconveniently complicating the harvest, was a direct result of the extended flowering period. Seed yield was closely related to the number of seeds per pod, and the single seed weight positively correlated with the endosperm yield. Increased soil water stress lead to reduced pericarp formation. Biennial alternations in reproductive growth were most common. "On"- respectively "off"-bearing status affected entire orchards, trees, or tree parts. Among the important triggers for alternate bearing were frost and increased soil moisture stress. Once initiated by crop loss, the cyclic bearing behaviour was maintained presumably due to endogenous constellations with both hormonal effects and competition for resources being involved. In "on"-years, developing fruits and seeds inhibited both vegetative growth and development of new flowers. Fruit characters varied in alternating trees according to the bearing status, inasmuch as in "on"-years, both fruit and single seed weight was reduced. Crop quality in carob is determined by the seed contents of the pod. From the present study it is concluded that there is a potential for an increase of both seed yield and endosperm yield by improving the ovule-to-seed ratio respectively by controlling excessive overload of trees. In order to enhance seed set a broad grafting program aiming at the increase of both density and genetic diversity of pollinators is suggested. Also, research on the causes for seed abortion and on pollinator properties, i.e. pollen quality and compatibility is required. Tree overload could be controlled by selective thinning of flowers aiming at creating an intra-tree equilibrium. Measures for qualitative crop improvement could give new incentives for all participants in the carob business. An essential prerequisite in this context, however, would be a future seed-yield-based remuneration for the carob farmers. It is furthermore recommended to look for alternative cultivars to partly substitute the strongly alternating 'Mulata', particularly for cultivation at marginal sites.
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Nineteen Tunisian carob populations from different sites were studied to assess their genetic variation based on pods and kernels measures. The mean of the main descriptive morphological values of pods were weight (16.39g), length (168.9mm), width (20.5mm), lateral thickness (8.3mm), central thickness (5.8mm), number of viable kernels (12.26), number of aborted kernels (1.38), kernels weight (0.2g), kernel length (9.1mm), kernel width (6.9mm), kernel thickness (4.1mm) and kernel yield (17.2%). Most of the parameters measured showed significant differences (P
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An in vitro propagation protocol based on axillary bud proliferation has been developed for mature female trees of Ceratonia siliqua L. `Galhosa' and `Mulata'. Browning and contaminants were the major obstacles for culture establishment. Shoot culture initiation was greatly influenced by explanting season, with the highest survival percentage observed in spring. The cultivar, cytokinin type and concentration were the most important factors affecting shoot multiplication. The best multiple-shoot response was obtained with `Mulata' on Murashige and Skoog medium supplemented with 4.44 M 6-benzyladenine or 4.56 M zeatin. Rooting was achieved on growth-regulator-free medium after basal dipping of shoots in indole-3-butyric acid (4.9 mM). Plantlets were successfully acclimatized (80–85%) under high relative humidity and then moved to the glasshouse. A field trial was established to follow their agronomic behaviour.
Microcuttings and stem fragments from an adult 8-year-old plant of Ricinodendron heudelotii were aseptized and cultured on a half strength Murashige and Skoog medium (MS/2). This paper analyses the impact of 6-benzylaminopurine (BAP) (1 to 5 mg/L) on budding and shoot development. With 4 mg/L of BAP, 100% of microcuttings sprout and give shoots after 52 days of culture. This study also examines the effect of kinetin (1 to 5 mg/L) on the proliferation of buds on microcuttings and on calli obtained from stem fragments, cultured on MS/2 supplemented with 5 to 7 mg/L of 2.4-dichlorophenoxy acetic acid (2.4-D). Eighty-six per cent of microcuttings form buds at 3 mg/L of kinetin with a mean of 8.2 buds per microcutting. A hundred per cent of calli form buds at 3.5 mg/L of kinetin with a mean of 5.6 buds per callus. The effect of naphthalene acetic acid (NAA) (1 to 5 mg/L) on rhizogenesis is also studied. At 1.5 mg/L, 85% of microcuttings with developed axillary buds give roots against 21% of buds isolated from microcuttings or calli and subcultured on the same rooting medium. Acclimatation of plantlets is successful in 53.4% of cases. Conditions defined in this work constitute a new method for producing plantlets of Ricinodendron heudelotii in view of the propagation and domestication of this species.
In this study, juvenile and 1 year-old apical and axillary buds collected from selected mature trees of cultivars from Italy, Portugal, Spain and Morocco, were used for in vitro initiation. Several methods of superficial disinfection were tested. Two media were used: MS (Murashige and Skoog, 1962) and 1/2MS (half-strength macronutrients MS) supplemented with 0.5 mg/L BA (6-benzyladenina). The lower rate of contaminations, associated with the best morphogenetic response of the buds, were obtained with explants from juvenile shoots collected between April and October treated with HgCl2 at low concentrations (0.1, 0.2%) for 5 min. Commercial bleach and ethanol were not efficient to get an effective response. Half-strength MS medium supplemented with 0.5 mg/L BA, was the best medium for the establishment of cultures since it did not cause hyperhydricity symptoms. On the whole the results show that the in vitro establishment of several carob tree cultivars is possible but adjustments are needed depending on the variety, explant type and season.