Effect of Cytokinins on the Micropropagation of Carob ( Ceratonia siliqua L.) through Shoot Tip Culture

In order to determine the most suitable cytokinin for the micropropagation of carob (Ceratonia siliqua L.), the effect of four cytokinins: BAP, zeatin, kinetin and 2-iP, was tested on explants derived from young seedlings of seven days. Organogenesis is better in the presence of BAP (0.5 mg/l), while buds growth is favored by zeatin (0.5 mg/l). The combination of the most suitable cytokinin (BAP at 0.5 mg/l) with IBA improves the caulogenesis when the concentration of this latter is low (0.1 mg/l); high concentrations of IBA have an inhibitory effect on elongation and neoformation of shoots and leaves. The multiplication and growth of shoots are more favorable on WPM medium in the presence of BAP (0.5 and 1 mg/l) alone or combined with 0.5 mg/l with GA 3 , while rooting is mainly favored by IBA, especially at 2 mg/l.


Introduction
This Carob (Ceratonia siliqua L.) is a Fabaceae Caesalpinioideae characteristic of Mediterranean regions and more or less equivalent climates. Generally spontaneous, it is currently cultivated on a large scale for its ecological and economic virtues. It is doing well in most Mediterranean climates despite their irregularity and on different types of soils [1] [2] [3] [4] [5].
These assets emerge the interest to control propagation techniques of this species by in vitro culture for the production of quality plants. 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. In addition, shoot tip culture is a simple and fast method for large-scale propagation of carob.

Explants Origin and Culture Conditions
Seeds come from mature fruits harvested in August from a vigorous tree in Bni Mtil region, at the edge of Tetouan Chefchaouen road (Morocco). Germination was carried out in flasks containing sterile agar-water (0.7%), after disinfection and scarification with 36 N sulfuric acid for one hour, followed by three successive rinses for 10 min with sterile distilled water. After one week, germination rate is close to 100%. Apices of young seedlings, whose size is 4 to 5 mm, were cut before epicotyl stage and directly transplanted into 200 ml flasks containing 50 ml of WPM culture medium [25], supplemented with Murashige and Skoog [26] micronutrients and vitamins, 30 g/l of sucrose, 0.1 g/l of myo-inositol and 0.7% of agar. pH was adjusted to 5.8 before autoclaving.
Explants were kept in a culture room with a photoperiod of 16

Effect of Different Cytokinins
Four cytokinins: BAP, zeatin, kinetin and 2-iP, at two concentrations: 0.5 and 1 mg/l were studied on WPM basal medium.

Effect of BAP-IBA Combination
In the first experiments, different concentrations of BAP (0.1, 0.3, 0.5, 1 and 1.5 mg/l) were combined with IBA (0.1 mg/l) on WPM medium. In the second step, the most favorable BAP concentration (0.5 mg/l) was selected and IBA concentration was varied (0.1, 0.2, 0.5 and 1 mg/l).

Multiplication and Elongation of Shoots Obtained from Apex Culture
In the first step, multiplication of shoots obtained from apex culture was accom-

Rooting
Shoots rooting after the multiplication phase was obtained after its induction in the dark by the auxins for one week on a half-diluted medium, composed of MS macronutrients, micronutrients and vitamins, sucrose (3%), myo-inositol (0.1 g/l) and agar (0.7%). Different auxins (IBA, IAA, NAA and 2,4-D) were tested at 1 and 2 mg/l. Shoots were then transferred to PGR free medium in the presence of light. Results were evaluated after 30 days.

Plantlets Acclimatization
After thirty days, vigorously rooted plantlets were transferred to pots containing peat and were sprayed with a 1% Benlate solution. Transparent plastic bags covering each plantlet ensured high humidity. Cultures were watered once a week with 1/2 MS mineral solution. They were kept in the culture room under the same conditions as before.

Effect of Cytokinins
Shoot size varies markedly according to cytokinin type and concentration. Zeatin significantly promotes growth in length at 0.5 mg/l, whatever the measurement period. The size of the shoots is a little weaker with 1 mg/l of zeatin and on the contrary minimal with 1 mg/l of BAP and 0.5 mg/l of kinetin (Table 1   Neoformation of shoots and leaves is favored by the BAP, significantly at 0.5 mg/l from the 30th day. Zeatin is in the second place, significantly at 0.5 mg/l. Kinetin and 2-iP are not very effective with more or less similar results to the control without PGR. As well, 2-iP favors undeveloped explants ( Table 2).

Effect of BAP-IBA Combination
In the presence of IBA (0.1 mg/l), shoots growth depends on BAP, but seems to be unaffected by its concentration: their size is between 14.8 and 16.7 mm and forms the same statistical unit for the five concentrations studied, whereas in the absence of BAP, it is only 12.4 mm. The combination BAP (0.5 mg/l) + IBA (0.1 mg/l) promotes shoots and leaves formation. Moving away from this concentration of BAP, the number of shoots and leaves decreases (Table 3, Figure 1(c)).
The combination of other AIB concentrations with BAP (0.5 mg/l) does not improve shoot growth or caulogenesis. The previously studied combination BAP (0.5 mg/l) + AIB (0.1 mg/l) remains statistically the most favorable for shoots a b c

Multiplication
Shoot growth is not strongly influenced by the choice between WPM and MS media. Shoots size and the number of leaves are significantly higher in 0.5 and 1 mg/l BAP compared to 1.5 mg/l. WPM medium tends to give more shoots than MS medium ( Table 5). The combination of GA 3 with BAP slightly increases shoots size compared to the control (without GA 3 ), especially at concentrations between 0.3 and 0.8 mg/l, which give similar values (Table 6, Figure 2(a)).
Shoots neoformation is inhibited by high and low concentrations of GA 3 ,

Elongation
Shoots elongation after one month is usually more remarkable between the second and third week. The combination BAP (0.1 mg/l) + IBA (0.1 mg/l) seems to be the most favorable for shoot elongation. It is the same for moderate concentrations of GA 3 (0.3 to 0.5 mg/l) combined with BAP (0.5 mg/l). However, it should be noted that shoot elongation tends to fade away as it approaches the fourth week ( Figure 2(b), Figure 3 and Figure 4).

Rooting
IBA was the most suitable auxin for rooting of shoots resulting from the multiplication by apex culture. 2 mg/l concentration gave a rooting percentage close to 90% and a satisfactory roots size (6 cm). Also, 1 mg/l gave good results, in particular for the neoformation of roots (6 per plantlet) and their size. However, IAA at 2 mg/l, NAA and 2,4-D do not favor rooting (Table 7, Figure 5(a)).

Acclimatization of Rooted Plantlets
Acclimatization of young plantlets after the multiplication phase was successful with a percentage of 80%. Sometimes, plantlets suffer leaf necrosis and wilt. After acclimatization and transfer to the soil, young plants are generally well maintained ( Figure 5(b)).

Discussion
The study of the effect of cytokinins on the culture of carob apex showed that a b American Journal of Plant Sciences    [28] [29].
Nevertheless, the neoformation of shoots and leaves is more favorable in the presence of BAP, especially at 0.5 mg/l, whatever the period of measurement, Compared with MS macronutrients, those of WPM allow better shoots and leaves formation, in agreement with previously published studies on carob [21] [23] [24]. The combination of BAP (0.5 mg/l) with different GA 3 concentrations improved slightly shoot size, while organogenic activity decreased a little, but BAP (0.5 mg/l) + GA 3 (0.5 mg/l) combination gives healthy shoots without physiological disorders and the use of BAP alone is sometimes accompanied by leaves yellowing and necrosis, especially at high concentrations. In reality, the combination of BAP at moderate concentrations with low concentrations of GA 3 has been used successfully [28] [32] [33] during shoot multiplication phase from various carob explants, while we reported negative effect of this combination on shoots multiplication from cotyledonary buds [29]. Consequently, propagation of carob shoots derived from apex is better on WPM medium in the presence of BAP (0.5 mg/l) alone or in combination with GA 3 (0.5 mg/l).
Shoots rooting after the propagation phase is more favorable in the presence of IBA, especially at 2 mg/l, whereas with the other auxins, rooting rate is lower.  [34] concluding that IBA at 2 mg/l is the ideal auxin for rooting.
Plantlets acclimatization on peat was sometimes confronted with some problems, such as shoots wilting and leaf necrosis. The acclimatized and periodically watered plantlets remain in good condition for ten to twelve months, but after this period, they begin to undergo foliar necrosis, hence the need to transfer them to the soil of a natural environment. After this, the acclimatized plantlets are well maintained and begin to bloom new buds after three weeks.

Conclusions
The study of the effect of cytokinins on the culture of carob apex showed that zeatin and BAP at low concentrations (0.5 mg/l) are the most favorable for shoots elongation, as well as for neoformation of shoots and leaves. In addition, initiation of the culture of carob apex can be easily established in the presence of moderate BAP concentration, close to 0.5 mg/l, alone or in combination with low concentrations of IBA (0.1 mg/l). Furthermore, WPM macronutrients allow better formation of shoots and leaves than those of MS and, the incorporation of BAP at moderate concentrations, combined with low concentrations of GA 3 , improved slightly shoots size and gives healthy shoots without physiological disorders. Moreover, the addition of 2 mg/l IBA alone to MS medium ensures the best shoots rooting. Finally, to achieve acclimatization, twelve-month-old acclimatized plantlets on peat must be transferred to the soil of a natural environment, in order to avoid shoots wilting and leaves necrosis.
The micropropagation of carob tree by apex culture is an effective tool for mass propagation and conservation of this important and rare medicinal plant in Morocco. Nevertheless, the choice of the optimal compositions of the culture media is essential to succeed in this protocol.