In Vitro Clonal Propagation from Juvenile and Different Explant Types of Two Edible Annonaceae Species: Annona muricata L. and Annona squamosa L.

Annona muricata L. and Annona squamosa L. are tropical species whose fleshy fruit is edible. They offer real possibilities for socio-economic use, particularly in the fields of medicine, nutrition, ecosystem conservation and the poverty alleviation. This study was set up to evaluate different methods of micropropagation from juvenile material for the regeneration of these species. Thus, MS medium supplemented with [BAP 2 mg·L −1 ] i.e. M2 produced 2.87 newly formed shoots from the cotyledonary nodes of A. muricata. For the terminal apices of A. squamosa, it was MMS medium supplemented with [BAP 2 mg·L −1 ] i.e. MM2 that was most conducive to new shoot formation (3.12). The addition of 0.1 and 0.2 mg·L −1 of NAA in the M2 medium, made it possible to have the best elongations and average number of nodes for the new shoots from


Introduction
Annona muricata L. and Annona squamosa L. are forest species of great socio-economic interest. Their fleshy and tasty fruits are very appreciated by the populations. Their bark, roots and leaves are also widely used in the African pharmacopoeia to treat various diseases such as sleep disorders, dermatoses, mycoses, diarrhea, etc. [1] [2]. In Senegal, these species are exploited in small agricultural gardens and orchards in the "Niayes" area, from Saint-Louis to the small marine coast. They are also cultivated in the southern regions as well as the groundnut basin in the Center of Senegal. They are part of the income-generating subsistence crops for farmers, especially during the lean season. However, their exploitation in the Sahel region has so far only been done through conventional methods such as seedling germination [3] [4]. However, due to its irregularity and slowness, this method is not sufficient to ensure good regeneration of these species [5]. Propagation by sowing seeds also leads to heterogeneous progeny, although the germination rate remains high for A. squamosa seeds [6]. This is just as valid for other methods such as suckering, grafting and cuttings, more rarely used, hence the need in Senegal as in the African regions where they are exploited, to have recourse to in vitro propagation methods. Thanks to this technique, the producer can obtain clonal plants from elite trees in sufficient quantity to satisfy the demand on the market and a continuous production independent of climatic hazards and seasons. In fact, unlike the conventional method of seed propagation, which gives a single individual per seed, the production of plants by in vitro culture makes it possible to obtain as many copies as desired from a single explant [7]. Despite its many advantages, the multiplication of Annonaceae by tissue culture techniques is almost non-existent in Africa and Senegal, in particular. Several works focusing mainly on the in vitro culture of Annona muricata L. and Annona squamosa L., have already been published in Latin America and Asia by various authors but with different methodologies and results. The articles published on these species mainly focus on the pharmacological properties of various chemical substances extracted from various organs.
In vitro vegetative propagation can be carried out either from material obtained from young plants germinated from the seeds or from adult material taken directly from elderly subjects. Nowadays, the preservation of the genetic diversity of living organisms has become a necessity [8], especially for species of great socio-economic interest.
In this context, a strategy was developed for in vitro vegetative propagation of determine the best elongation, multiplication, and rooting media; 4) and, define finally the best acclimatization conditions for the new plants obtained.

Plant Material
For each species, 3 types of explants measuring 1 to 2 cm long and taken from sterile 30-day-old seedlings were tested. These were terminal apices, axillary and cotyledonary nodes. These different types of explants are transferred separately and individually into sterile glass culture tubes (150 × 25 mm) filled up with 20 mL of solidified culture medium.

Culture Media
The basic nutrient medium used for A. muricata is the complete medium of Murashige & Skoog [9], while for the explants of A. squamosa, the basal one is MMS medium; it corresponds to the modified Murashige & Skoog medium [10]. These media were solidified with 8 g·L −1 agar at pH 5.7 and contained 30 g·L −1 of sucrose. Growth regulators were added or not into the different media. The composition of the different hormones tested alone or in combination in the different culture media is given in Table 1. The same culture media have been tested in the presence of 2 g·L −1 of activated charcoal. The hormonal treatments were multiplied by a factor of 10 due to the adsorbing action of the activated charcoal on phytohormones. The second experiment included 3 other culture media. This is MS + BAP 2 mg·L −1 to which NAA has been added at concentrations of 0.1, 0.2 and 0.5 mg·L −1 . All media were dispensed into culture tubes (150 × 25 mm), i.e. 20 mL per tube, then sterilized by autoclaving at 110˚C for 20 min.

Experimental Set-Up
For each type of explant, a number of 36 per medium and per species was evaluated. In other words, three replicates of 12 test tubes were used for each treatment. The tubes are first incubated in a culture chamber at 27˚C ± 1˚C in the dark for 5 days, then under a 16 h day/8h night photoperiod and an incident light of 4000 Lux.
Measurements on each type of explant were performed after 30 days of incubation. The parameters measured concerned the presence or absence of resumption of activity (reactivity rate), the number and length of newly formed shoots and the number of nodes. Thus, from these data, the means were calculated, the coefficients or multiplication rates determined and the best regeneration media deduced.  (Mathur et al., 1995); BAP: 6-Benzy-laminopurine; KIN: Kinetin (6-furfuryl aminopurine); NAA: 1-Naphthaleneacetic Acid.

Rooting Procedure
Newly formed third generation of shoots, i.e. resulting from three successive subcultures lasting thirty days each, are induced in the dark in the MS/2 medium; the macro-elements of which have been diluted by half, supplemented with IBA used alone or at respective concentrations of 25 mg·L −1 and 50 mg·L −1 .
These media contained 2 g·L −1 of activated charcoal, 20 g·L −1 of sucrose and were solidified with an 8 g·L −1 agar. The pH was adjusted to 5.7, according to the method applied by Farooq et al. [11] and Ba et al. [12]. The explants were induced for a duration of 1, 3, 5 days before being transferred to the incident light in the MS(0)/2 expression medium i.e. without hormones. For each vitroplant issued from each type of explant and each treatment, a number of 36 explants was used for each duration and induction medium. A batch of 12 explants was maintained as a control batch, without root induction, in the MS(0)/2 medium.
After 30 days, measurements were taken to determine the rooting rate, the number of newly formed roots per explant and the length of the roots, for each treatment.

Acclimatization
After 4 weeks of incubation in the expression medium, the well-rooted young plants are transferred to weaning conditions according to the procedure of Ba et al. [12].

Statistical Analysis
The different treatments were discriminated by multiple comparison of the means after analysis of variance followed by the Student-Newman-Keuls test at the probability threshold of 5% (SPSS 19.0 software). for an average length of 5.16 cm ( Table 2). 2) Annona squamosa -Apices: The best recovery rate was observed in MM4 medium [MMS + KIN

Effects of NAA Combined to BAP on the Morphogenesis of Juvenile Explants
The results related to the morphogenesis of the explants for the two Annona species are reported in Table 3 and Plate 1.    (Table 3)

Effect of Induction with IBA for 1, 3 and 5 Days on the Vitroplant Rooting
Referring to      (Table   4).  Table 4).

Annona squamosa
The best rooting rate (66.67%) was also obtained for vitroplants from cotyledonary nodes thanks to a 3-day-induction period, with 3.37 roots and length of  (Table 4).

Acclimatization
The different types of explants are transplanted into cups containing a sterile sand-soil mixture and then stored in a mini-greenhouse with the shutter completely closed for a week. This process kept the plants produced in vitro in an atmosphere of high relative humidity, but the mini-greenhouse is fitted with a plexiglass bell with an adjustable opening.

Influence of Growth Regulators on the in Vitro Morphogenesis of Different Types of Explants
One week after the first in vitro introduction of the different explants, an onset of browning due to the presence of polyphenols was noted. This could be due to the exposure of seedlings resulting from germination to light which would promote the synthesis of the polyphenols. According to [14], roots of young plants grown under in vitro light conditions produced more polyphenols than leaves of adult plants during the fall. The authors [15] have already reported browning of the material in A. muricata when it was 16 to 24 days old, which has a negative impact on its viability. Furthermore, abiotic stress such as temperature extremes, drought, flooding, largely influence plant development and crop productivity [16]. It is well known that the biosynthesis of phenolic compounds is generally activated by certain abiotic factors such as extreme temperatures, salinity, pollution by heavy metals, light, especially ultraviolet radiations [17] [18]. As a result, the explants of the two species were transferred to the different culture media in which 2 g·L −1 of activated charcoal were incorporated.
After 6 days of culture, the outlines of newly formed shoots were visible at the level of the cotyledonary and axillary nodes and were more numerous in the media enriched with hormones for explants of A. muricata. For the apices of A. squamosa, they started to appear from the 8 th day. New shoot formation took place in the area where the cotyledonary leaves were inserted. Indeed, the growth and development of plants can be modulated by the synergistic contributions of auxins and cytokinins [19]. The authors [20] had already shown that the organogenic potential of the cotyledonary nodes of Vigna mungo is strongly influenced by the type of growth regulator, the growth and the hormonal combination used. The organogenic properties of cytokinins were explained, in part, by their interactions with the sucrose present in the culture medium [21].
For the 2 species studied, the BAP used alone or in combination with Kinetin was found to be more effective than the control medium or Kinetin employed alone for the new growth of shoots and the number of nodes. Kinetin alone had a better effect on elongation than BAP even though this allowed for better bud breakout. The same result was obtained by [22] in Ferronia limonia. According to [23], BAP was more effective than Kinetin for regeneration and shoot elongation in Parkia biglobosa. The same observation was made by [24]) on Balanites aegyptiaca and [25] on Vigna radiata. The work of [26] on Pentadesma butyraceae Sabine shows that BAP promotes resumption of apices activity. These authors [27] showed that the best medium for the propagation of Annona annua shoots is MS medium supplemented with 1 mg·L −1 of BAP. The incorporation of BAP into the culture medium generally improves the regeneration of explants as reported by [25]. On the other hand, Kinetin essentially promotes the elongation of buds [28]. It is noted that an increase in the cytokinin content is accompanied by a reduction in shoot elongation and the average number of nodes in A. squ- amosa while in A. muricata explants, BAP at 5 mg·L −1 allowed an elongation of the newly formed shoots and an increase in the number of nodes, but only for the cotyledonary nodes. According to [29], a cytokinin concentration greater than 2 mg·L −1 has a detrimental effect on multiplication and rooting in most Annonaceae. Thus, [30] used cytokinin concentrations not exceeding 1 mg·L −1 for the in vitro multiplication of Annona glabra. This action of high doses of cytokinin on the in vitro morphogenesis of Annonaceae species seems to confirm the observations of [28] on Vigna unguiculata. High concentrations of BAP would favor apical dominance in Ixora margaretae [31] and in Morinda sp. nov A [32].
The work of [33] demonstrated that the addition of BAP in the culture medium would induce the apical dominance during in vitro culture of the apical part of Mentha spicata H. (mint) and would, therefore, intensify the height growth of the plant. Furthermore, [34] had noticed in Annona glabra that the BAP concentrations greater than 0.5 mg·L −1 , added in a medium without activated charcoal, caused a strong callogenesis and a vitrification of the explants. This ultimately causes necrosis of the tip of the stems and leaf abscission that we observed in A. muricata and A. squamosa. These physiological disorders are frequently mentioned during in vitro culture of other Annona species [35]; they have been associated with calcium deficiency [36] or ethylene accumulation [37] in A. squamosa. The lack of calcium considerably affects cell division at the level of the meristematic zone as well as the synthesis of pectin [38]. However, [39] have already shown that a high concentration of cytokinins affects the accumulation and use of calcium by the vitroplant during the micropropagation of Annonaceae. Increasing cytokinin concentration induces a decrease in the rate of explant regeneration [40]. In addition, at high doses, cytokinins promote the accumulation of iron in many fruit species of the Annonaceae family, which promotes oxidation during in vitro multiplication [30]. However, the type of vial used and the sealing technique adopted are factors responsible for the development of these disorders; they would lead to maintaining a high relative humidity of the air inside the container, preventing the transpiration of the plant necessary for calcium to reach the xylem of the plant, and they would make gas exchange with the outside atmosphere impossible, and would, thus, facilitate the accumulation of gases produced by the tissues at physiologically active levels [35].
The addition of NAA to the culture media together with the cytokinins did not increase the number of newly formed shoots compared to the cytokinins used alone for the 2 Annona species. However, the combination of BAP with NAA appears to be more efficient in stimulating shoot elongation and increasing the number of A. muricata nodes. This favorable action of the cytokinin-auxin combination on the morphogenesis of young material is mentioned by [41] on Santolina canescens, [42] on Bupleurum fruticosum, [24] [46]. However, in some cases, NAA can promote shoot proliferation for many species of Hyacinthaceae [47] and Saussurea lappa [48]. On Plantago lanceolata, [49] obtained good regeneration of hypocotyl and cotyledon explant shoots in MS medium supplemented with BAP (0.75 mg·L −1 ) and NAA (0.2 mg·L −1 ).
The NAA-BAP combination, however, caused callus formation at the base of explants in vitro cultured for many explants. This callogenesis has already been reported by [50] on Nigella sativa, [25] on Vigna unguiculata and [51] on Nigella damascena. The intensity of callus formation increases with increasing NAA concentration in the growing medium, which significantly limits shoot regeneration in Vigna unguiculata [52]. According to [53], it is frequently observed in species with marked apical dominance. It is due to the accumulation of auxin at the base of the apical explant [54]. The auxin, in the presence of cytokinins, would stimulate the proliferation of cells located in the area of injury of the explant. The presence of callus constitutes a factor limiting rhizogenesis. Indeed, after one month of culture, one observed on certain explants a browning of the calluses which gives a bad surface of contact of the explant with the culture medium and seems, therefore, to slow down its growth.

Rooting
In A. muricata, the best rooting rate is obtained with vitroplants newly formed from axillary (41.67%) and cotyledonary nodes ( This stress would induce the production of ethylene, the concentration of which would tend to increase in confinement, which also stimulates the production of auxin. On the contrary, [61] and [62] demonstrated that the decrease in sugar concentration and the presence of agar favored rhizogenesis.
We also noted that the rooting rate remains quite high, especially for A. muricata for which the best rooting rate was 83.33%. For those of A. squamosa, this rooting rate was equal to 66.67%. The roots were, in general, more developed at the level of vitroplants resulting from the cotyledonary nodes where more lateral ramifications were observed for the 2 species. Furthermore, [63] could not exceed 50% rooting in the giant sequoia. This can be explained, by the fact that rooting is often more difficult in woody plants than in herbaceous plants [64].
The high rooting rates may be due to the combined beneficial effects of the basal medium used (MS/2), dark incubation, inclusion of activated charcoal, and induction with high concentrations of auxin. The authors [65] as well as [66] noted the importance of auxins in improving root system development. The author [67] reported that halving macronutrients facilitated rooting in many species. This effect is thought to be due to the reduction in the quantity of nitrogen in the medium [68]. The beneficial effect of MS/2 medium on rhizogenesis has also been obtained on avocado [69]. On the other hand, [70] showed that incubation in the dark, in the presence of IBA, increased the rooting rate of Pistacia vera vitroplants. This beneficial effect of darkness on rooting has also been observed on apple [71] and on Quercus rubra [72]. Beyond its action on polyphenols, the production and oxidation of which it inhibits [73], darkness increases the number of cells competent to induce the appearance and development of roots thanks to the etiolation [74] and, thus, avoids the destruction of the growth regulators included in the medium by reducing the peroxidase activity [72]. According to [75], 5 days incubation in the dark allowed root inducing cells to differentiate on Quercus robur. However, in this species, during incubation in the dark, there is senescence and necrosis of the shoots [76]. According to [72], activated charcoal not only stimulates root development, but at the same time, adsorbs excess growth regulators. It also acts to prevent the harmful action of polyphenols and helps to obscure the environment of the explant which is favorable to rhizogenic induction [77] [78].

Acclimatization
The low survival rate during the acclimatization of young plants resulting from O. Ba et al. in vitro propagation is one of the factors limiting this technique for the economic exploitation of many species of economic importance [79].
Acclimatization in a mini-greenhouse followed by gradual contact with the surrounding environment from the first week onwards made it possible to obtain, on A. muricata, 83.33% of young plants from the apices which survived, while for those from axillary and cotyledonary nodes, the survival rate was 75%.
For A. squamosa, the survival rates were, respectively, 75%, 83.33% and 66.67% for young plants from the apices, cotyledonary and axillary nodes. Similar results have been reported by [80] on Carob (Ceratonia siliqua L.) microplants gradually acclimatized to the ambient atmosphere. Confining the young plants in an atmosphere saturated with humidity (90% to 100%) at the start of weaning i.e. close to in vitro conditions at a high temperature, has been shown to be beneficial for their successful acclimatization. Indeed, according to [24] plants resulting from in vitro culture generally have a thinner cuticle than that of mother plants, which causes their rapid desiccation when the relative humidity is lowered rapidly by the passage ex vitro. Thus, [81] noted that 43% of plants of Prosopis juliflora and P. chilensis die off during acclimatization. On the other hand, the very high relative humidity inside the tubes during in vitro cultivation causes the leaves of young plants to have a very high amount of non-functional stomata compared to those of adult plants adapted to in vivo conditions [82], [83]. Gradual weaning in a mini-greenhouse, therefore, allows plants to acquire anatomical, physiological and metabolic characteristics that allow them to better adapt and survive during their transplantation under natural conditions.

Conclusion
This work has shown the importance of hormonal combinations in the micropropagation of these Annonaceae species. It is important to note the beneficial effects of BAP in new shoot formation and its positive synergistic effect with NAA in shoot elongation, but the effect of these hormones varies depending on the type of juvenile explan tintroduced in vitro. Among the explants, the cotyledonary nodes are the most reactive for the neoformation of shoots. It also seems that IBA is better than NAA for the rhizogenous induction of vitroplants and improves their branching in lateral roots. The gradual weaning of the vitroplants in a mini-greenhouse, with an adjustable opening, is more favorable to the acclimatization of the plants ex vitro whatever the initial origin of the explants.