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.

Referring to Table 4, depending on the IBA concentration and the induction time, for each species, the results were as follows:

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. Watering is carried out every 2 days with Hoagland and Arnon’s nutritive solution (1938) for the first 2 weeks, then with tap water. On the 12^th day of acclimatization, for A. muricata, a decay rate of 8.33% for the explants resulting from the axillary and cotyledonary nodes was noted. For A. squamosa, it was 16.67% for those originated from the apices and axillary nodes, while for plantlets from cotyledonary nodes, it was 8.33%. From the 14^th day, the shutter is opened halfway to avoid prolonged confinement. At the 21^st day, the plexigas bell is fully opened. The plantlets were first maintained in the shade under the bench for 3 days and then placed on the bench. Thus, on the 30^th day, the survival rates were 75% for the plants issued from the cotyledonary and axillary nodes while for those from the apices of A. muricata, 83.33% survived. For the plants of A. squamosa from the apices, axillary and cotyledonary nodes, the survival rates were, respectively, 75%, 66.67% and 83.33% (Table 5, Plate 2).

APX: Apex; AN: Axillary Node; CN: Cotyledonary Node. In the same column and for the same type of explant, the numbers followed by the same letter are not significantly different at the 5% threshold of the Newman-Keuls test.

Plate 2. Young plants resulting from the juvenile material after 30 days of weaning under shade. A: Annona muricata; B: A. squamosa; A1, B1: Plants from the apices; A2, B2: Plants from axillary nodes; A3, B3: Plants from cotyledonary nodes.

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. squamosa 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] on Balanites aegyptiaca and [23] on Parkia biglobosa. The beneficial action of NAA on shoot elongation has already been reported by several authors [32] [43] [44]. Other authors [45] claimed the opposite, NAA would greatly affect bud formation in Plumbago rosae. Several studies reported that the BAP-NAA combination induces a decrease in the number of regenerated shoots [20] [25] [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.

In A. muricata, the best rooting rate is obtained with vitroplants newly formed from axillary (41.67%) and cotyledonary nodes (83.33%) following a 5 day-rhizogenic induction in an MS/2 medium. Supplemented with [IBA 50 mg·L⁻¹] and in the presence of 2 g·L⁻¹ of activated charcoal while an induction of 24 h makes it possible to have a rate of 41.67% for the vitroplants newly formed from the apices. In A. squamosa, the best rooting rates are obtained for vitroplants from the apices (41.67%) and those from the cotyledonary nodes (66.67%) following a 3 day-rhizogenic induction with [IBA 50 mg·L⁻¹] whereas for vitroplants from axillary nodes, this rate is obtained following a 5-day-induction with [IBA 25 mg·L⁻¹]. The rooting of woody species is generally dependent on hormonal treatment as pointed out by [23] in Parkia biglobosa. The authors [55] also noted an 80% rooting rate on Acacia tortilis subsp. raddiana after treatment with IBA compared to NAA. IBA would, therefore, be more conducive to the rhizogenic induction of woody species, the latter being difficult to root in vitro [22] [56] [57]. In addition, [27] obtained better rhizogenesis in Artemisia hololeuca in the presence of IBA compared to the use of NAA.

The consistency of the rooting medium, like the high concentrations of auxin, also seems to strongly influence the appearance of callus at the base of explants as with explants of Annona muricata and A. squamosa in the presence of high concentrations of IBA. Likewise, [58] found that rooting of apple tree can be achieved in a solid or liquid medium by using vermiculite with better results in the latter case. The authors [59] used a solid and liquid medium with paper and vermiculite support for the M106 apple tree rootstock and obtain an excessive development of the callus, i.e. 40% of rooting in the solid medium while the use of liquid medium increases the rooting rate to 80% and results in a noticeable reduction in callus development. This significant difference is explained by the fact that nutrients are more readily available for the explant [60]. In addition, the agar would create a critical turgor pressure which puts the cells under stress. 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].

The low survival rate during the acclimatization of young plants resulting from 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.