Callus Induction from Carob (Ceratonia siliqua L.) Seedlings and Leaves of Mature Tree

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); Original Research Article El Bouzdoudi et al.; ARRB, 19(2): 1-13, 2017; Article no.ARRB.37037 2  Hypocotyls taken from 7-day-old seedlings (55% success rate on WPM containing 2.22 μM BAP and 5 μM NAA);  Differentiated leaves taken from mature tree (84% success rate on WPM medium, containing 4.44 μM of BA and 2.26 μM of NAA). In general, production of primary calli and their growth after transplantation was better on WPM medium supplemented with 2.5 μM NAA and 2.22 μM BAP.


INTRODUCTION
The carob tree (Ceratonia siliqua L.) is a longlived evergreen and thermophilous tree thriving in habitats with mild Mediterranean climates. It grows well in warm temperate and subtropical areas, and tolerates hot and humid coastal areas [1]. It is an important aromatic and medicinal plant that belongs to the family of Leguminosae and is currently considered among the most prominent fruit and forest trees existing in Morocco.
Due to its particular biological and agroecological features, such as resistance to salinity, adaptation to poor soils, and minimal cultural requirements, the carob tree was included in the national list of priority as forest resources for conservation in Morocco. This species is used in reforestation actions and its cultivation in modern orchards is being undertaken to value marginal lands and substitute for drought sensitive species [2].
The presence of wild trees growing adjacent to established orchards, as well as the great variation in sexuality of different carob varieties (male, female, hermaphrodite and polygamous inflorescences) causes great intraspecific variability and a large number of cultivars [3]. The high phenotypic variability within and between cultivars has important implications for selection, cultivation practices, and establishment of new plantations and productivity optimization of this crop [1].
Traditional carob propagation has been achieved by grafting saplings with female buds of chosen productive trees [1]. This traditional method of propagation has failed to meet the market demand for new, selected plant material. Thus, the use of micropropagation techniques seems to be appropriate, in order to fulfill the increased demand for propagating this tree [4]. Callus formation may be a useful tool to establish growth control and to study metabolism of forest tree species [5][6][7]. It can also lead to the differentiation of organs and even plants whose origin can be easily explored anatomically and experimentally in a sterile and simple nutritive culture [8][9][10][11].
The aim of the present study was to optimize an easy and reliable method for rapid propagation of induced callus from different parts of carob seedlings and leaves of mature trees.

Preparation of explants
Mature carob pods were collected between August and September (2016) from a female tree, located at 30 km along the Tetouan-Chefchaoun road in the Amtil region, Western Rif, Morocco. The harvested pods were shelled and the recovered seeds were disinfected with sulfuric acid (H 2 SO 4 , 36N) for 60 min, followed by three rinses with sterile distilled water for 10 min each [33].
The disinfected seeds (30 seeds) were placed in 100 ml flasks, containing 50 ml of sterile distilled water and kept under stirring (10 rpm) for 48 h in a rotary shaker.
The seeds were then germinated in 200 ml flasks containing 50 ml of agar water (0.7%, pH = 5.8) previously sterilized (20 min at 120°C). Cultures were placed in an air-conditioned room at 25°C, illuminated by fluorescent tubes (Philips, 40W) having an intensity of 800 lux. The photoperiod was 16 hours of light per day and the relative humidity was 85%.
Apexes were collected from seven-day-old seedlings at the stage of two cotyledonary leaves, with a size of 4-5 mm.

Effect of auxins
The study of callogenesis in the presence of different solutions showed that WPM medium is the most favorable to the development of calli. Thus, in order to determine the most suitable auxin for the induction of calli, the effect of four auxins was tested: indole 3-acetic acid (IAA), indole-3-butyric acid (IBA), NAA and 2,4dichlorophenoxyacetic acid (2,4-D). For each, four concentrations were tested: 0.5; 2.5; 5 and 7.5 μM in combination with 4.44 μM BAP.

Callogenesis from Other Explants of Young Seedlings Grown In vitro
Carob tree explants were very favorable for callus induction. Any organ could be used. Among these organs, cotyledonary leaves and hypocotyls from seven-day-old seedlings were tested. Embryonic cotyledons derived from immature fruit seeds harvested in June were also used, after disinfection according to the protocol of El Bouzdoudi et al. [37]. The base medium previously used was preserved, supplemented with 5 μM NAA and 2.22 and 4.44 μM BAP.

Preparation of explants
Disinfection was carried out according to the protocol of Rolano et al. [38] with a 7% (w/v) filtered solution of calcium hypochlorite (Ca(ClO) 2 ), containing few drops of Tween 80 for 20 min, followed by rinsing with sterile distilled water for 5 min, then with 0.1% (w/v) mercuric chloride (HgCl 2 ) solution for 5 min. HgCl 2 is then removed by three successive rinses for 10, 10 and 15 min in sterile distilled water.
The leaves used are those of the year, not very sclerified. They are placed in 200 ml flasks containing 50 ml of nutrient solution.

Effect of macronutrients solutions
In order to test the impact of WPM and MS macronutrients, two concentrations of BAP (2.22 and 4.44 μM) were each combined with 2.26 μM ANA.

Effect of different auxins
As WPM solution was the most favorable for callogenesis, it was preserved to study the effect of six auxins (IAA, IBA, β-naphthoxyacetic acid

Effect of different auxins on the growth of calli
The calli obtained from the culture of apex and cotyledonary leaves were transplanted onto the same base medium in the presence of NAA at 5 μM combined with the BAP at 2.22 μM, seven times during 21 days. After their stabilization, the calli were cultured in the presence of four auxins (IAA, IBA, NAA and 2,4-D) associated with the BAP (2.22 μM). Two concentrations were chosen for each auxin (2.5 and 5 μM). After 21 days of culture and separation of the agar, the diameter of the callus and its fresh weight were measured. Each callus was packaged in aluminum foil and placed in an oven at 75°C. After 15 days of dehydration, their dry weight was measured.

Effect of NAA combined with BAP on calli growth
As the combination of 2.5 μM of NAA and 2.22 μM of BAP was the best for callus growth, it was retained for the study of callus growth over time for 45 days. Every five days, the diameter and fresh weight of a sample of about twenty calli was determined, and the dry weight was estimated after 15 days of dehydration.

Culture Conditions
Cultures were placed in the culture room at an illumination of 4000 lux and a temperature of 23-25°C during the day and 20°C at night. The programmed photoperiod corresponded to 16 hours of light per day. Results were read after one month for callus initiation and 21 days for their multiplication.

Statistical Analysis
For all the studies conducted, thirty replicates per treatment were taken and and the experiments were repeated three times. Statistical analysis to find differences among treatments was done by one-way ANOVA using SPSS ver. 16 (SPSS Inc., Chicago, USA). The significance of difference among means was carried out using Duncan's multiple range tests at P = .05 and the results are expressed as mean ± SE (standard deviation).

Effect of macronutrients solutions
Apex culture is accompanied by the formation of a callus that size varies according to the conditions studied.

Effect of auxins
Callogenesis from apex depends on the type of auxins and their concentrations. The NAA appears to be the most favorable for the development of calli. Indeed, the percentage of callogenic explants is maximum (90.33%), as well as the diameter of callus (14.1 mm). The 2,4-D is in second place with a percentage of around 76.30%, followed by IBA and finally IAA. The concentrations studied show that for the same auxin and from 2.5 μM, the results obtained are often similar and without significant difference. However, it should be noted that the status and color of calli are better only in the presence of NAA (Table 2).  (Table 3).

Callogenesis from Other Explants of Young Seedlings Grown In vitro
For carob, callogenesis can be induced from various explants. In most cases, callus development occurs along the organ in contact with the culture medium. The best percentage of callogenesis is obtained during the culture of embryonic cotyledons, with similar values for the two concentrations studied. However, calli size is larger in the case of cotyledonary leaves ( Table  4, Fig. 1d, 1e, 1f).

Effect of macronutrient solutions
The percentage of callogenesis as well as the diameter of the calli are greater on WPM medium and are respectively 84.00% and 10.6 mm for 4.44 μM of BAP; in this case, calli are generally in good condition and easy to transplant. The MS macronutrient solution is less efficient ( Table 5, Fig. 1g, 1h).

Effect of different auxins
The study of the impact of different auxins (IAA, IBA, BNOA, AIP, NAA and 2,4-D) associated with BAP (2.22 and 4.44 μM) on callogenesis from leaves of an elite tree, showed that NAA is the most effective for the induction of calli and their development, whatever the concentration of BAP. Percentages of callogenesis (78 and 84%) and callus diameter (10.5 and 10.7 mm) form the same statistical class. 2,4-D is in second place with good results close to those obtained with NAA, while other auxins are less effective, especially AIP and BNOA ( Table 6, Fig. 1i, 1j).

Effect of different auxins on calli growth
The transplanting of the calli under different conditions is accompanied by very considerable growth. In most cases, their size exceeds 20 mm after 21 days. Maximum values of size, weight of fresh and dry matter are obtained in the presence of NAA, in particular at 5 μM (28.1 mm, 4.13 and 0.40 g, respectively). The results obtained with the IBA come in the second place, but this time with 2.5 μM. Generally, IAA and 2,4-D give results with no significant difference ( Table 7, Fig. 2a to 2d).

Effect of NAA combined with BAP on calli growth
According to Table 8 and Fig. 3 and 4, it can be seen that during the first days, calli are less reactive, but as early as the second week, they are organized and the cell multiplication intensifies. Often, clubs are formed which eventually come together. The growth of calli is the most important between the fifth and fifteenth days. It results in a steep slope which tends to dampen on the twentieth day. Beyond this time, there is a marked slowdown in growth and a plateau is achieved. After the third week, calli begin to blacken. From the 45th day, their transplanting becomes very difficult. Measurement of the size and weight of fresh material are good parameters for visualizing the most active period (Fig. 2e to 2h).

DISCUSSION
For carob tree, callogenesis induction from various explants presents no problem. WPM medium is the most favorable among the three culture media studied for the initiation of calli. These results concur with those obtained in cork oak by El Kbiach et al. [39]. The low efficiency of SH and MS media could be attributed to their nitrogen richness, in particular ammonia nitrogen, which reduces the rate of cell proliferation [40,41]. Thus, WPM medium constitutes a medium of choice for the in vitro culture of carob tree.
The study of the effect of different auxins combined with BAP on callogenesis from apex shows that NAA is the most suitable for the development of calli, their growth and their maintenance. They are usually green yellow (chlorophylls) and easy to handle. Among the various other explants (cotyledonary leaves, embryonic cotyledons and hypocotyls), embryonic cotyledons also show a high callogenous capacity, but it remains slightly lower than that of the apexes.
The efficiency of NAA on callogenesis was proven by various researchers for other woody plants such as Quercus rubra [42,43], Q. robur [44] or Q. suber [39]. For carob, NAA was successfully used for callogenesis from hypocotyls (75%) and cotyledons (57.1%) with 1 mg/l NAA and 2 mg/l BAP [12], apex of young seedlings (66-80%) in the presence of 1 mg/l BAP associated with 0.1 mg/l NAA [15], also with nodal segments of young seedlings in a media containing 1 mg/l BAP in combination with 1 mg/l NAA [19]. However, for other studies on carob, the best results for callus induction were obtained with 2,4-D in the case of roots (28.6%) [12], anthers (100%) [29], immature seeds (67%) [30], portions of hypocotyls (58.3%) and cotyledons (100%) [18] and mature cotyledons of seed (100%) [31].    Among the concentrations of BAP combined with NAA, it was found that concentrations 2.22 and ar results without significant difference, in particular when the concentration of NAA is equivalent to 5 μM. The combination of 2.22 μM of BAP and 5 μM of NAA could therefore be a prerequisite for further studies. For Belaizi et al. [14], BAP ns higher than 0.5 mg/l result in high callogenesis. Carimi et al. [28] obtained good calli (16%) from ovules derived from young carob pods with 13.3 μM BAP, but they worked on MS medium supplemented with malt extract.
The leaves of the mature tree have a considerable callogenous capacity, in particular on the WPM medium supplemented with BAP (4.44 μM) and NAA (1.26 μM). The association of BAP with other auxins is generally less efficient, but 2,4-D has also considerable capacity. 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 observed by other researchers [4,13, The transplanting of calli obtained from different types of explants makes it possible to stabilize their proliferation. Overall, the conditions that favor this proliferation are those considered favorable for their initiation. The study of the effect of different combinations of BAP with auxins also showed that NAA is the most suitable auxin for proliferation of callus. The dry weights are similar for the two combinations studied (BAP at 2.22 μM and NAA at 2.5 or 5 μM). During the proliferation phase, we did not obser ; Article no.ARRB.37037 Fig. 3. Variation of the size of calli after transplanting on WPM medium, supplemented with 4. Variation of callus weight after transplanting onto WPM medium, supplemented with D has also considerable callogenous capacity. 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].
nting of calli obtained from different types of explants makes it possible to stabilize their proliferation. Overall, the conditions that favor this proliferation are those considered favorable for their initiation. The study of the binations of BAP with auxins also showed that NAA is the most suitable auxin for proliferation of callus. The dry weights are similar for the two combinations studied (BAP at 2.22 μM and NAA at 2.5 or 5 μM). During the proliferation phase, we did not observe primary somatic embryogenesis. Contrariwise, Custodio et al. [29], Ksia et al. [30] and Lozzi et al. [31] induced primary somatic embryogenesis from the obtained calli.
It should be noted that, after several successive subcultures, very friable calli clusters are obtained which can well serve to obtain cell suspensions. Tests in this direction are under study.
The proliferation of calli and their maintenance is blocked by the problem of blackening, which is accentuated by their age. From the third week, the calli begin to change color. After 40 to 45 days, they become hard and compact and their transplanting then becomes impossible. The blackening of calli may be attributed to an accumulation of polyphenols. Research on these polyphenols and their degree of accumulation depending on culture conditions would be useful.

CONCLUSION
The study of calli induction by using varied explants made it possible to optimize the conditions of callogenesis as well as those of their maintenance and their multiplication. WPM medium supplemented with BAP (2.22 μM) associated to NAA (5 μM) is the most favorable condition. Percentage callus formation in apex seedlings (89%) and embryonic cotyledons of seed (81%) was greater than in leaves of mature tree (78%), cotyledonary leaves (60%) and hypocotyls (54%) seedlings. Optimization of callogenesis can be exploited to initiate the culture of cell suspensions and to initiate somatic embryogenesis.