Many neglected and underutilized wild species (NUS) are nutritionally rich (Ghane et al., 2010; Johns and Eyzaguirre, 2006). Therefore, their erosion can have immediate consequences on the nutritional status and food security of the poor and their enhanced use can bring about better nutrition and fight hunger. Even though the link between agrobiodiversity and diet diversity is not automatic (Burchi et al., 2011), it is agreeable that the diminution of agrobiodiversity, to some extent, places considerable strain on the ease with which households are able to enjoy diversified, balanced diets. Awareness of the importance and value of crop wild relatives and of the need to conserve them has increased one of such high value plant that is in dire need for conservation, Vitex doniana Sweet (Black plum). It had been considered to belong to Verbenaceae family by different authors but in recent works, it has been transferred into Lamiaceae based on different evidences (Wagstaff et al., 1998). It is the most abundant and widespread Vitex species in Africa (Orwa et al., 2009). Black plum (V. doniana) is an indigenous species important for the livelihoods of rural populations in Benin Republic in particular and West Africa in general (Codjia et al., 2003). The fruits and leaves are the edible part of the trees. They are either eaten raw or after processing. The plant is also widely used in traditional system for medicinal purposes. The leaves, fruits, roots, barks and seed of the plant have been used as medication for liver disease, anodyne, stiffness, leprosy, backache, hemiplegia, conjunctivity, rash, measles, rachitis, febrifuge, as tonic galactagogue to aid milk production in lactating mothers, sedative, digestive regulator, treatment of eye troubles and kidney troubles. It has also been used for treatment of conditions such as infertility, anemia, jaundice, dysentery, gonorrhea, headaches, diabetes, chickenpox, rash and fever (Louppe et al., 2008; Orwa et al., 2009). Despite the widely known nutritional, medicinal and economic uses of V. doniana products, the species is still under-utilized, unimproved. To date, the species has been chosen as a model species to be domesticated in Benin (Codjia et al., 2003; Dadjo et al., 2012).The strong anthropic pressure affecting this species has caused its numbers to fall increasingly in its natural environment (Achigan-Dako et al., 2010). The planting of seedlings is negligible and the seeds of this tree have a very weak germinating capacity (Thies 1995). Sanoussi et al. (2012) reported that macropropagation rate of this tree species by stem cuttings is slow. Therefore, there is need to seek for alternative propagation methods. Tissue culture methods are considered as the most promising means to protect and propagate tree species of economic interest. In vitro propagation through somatic embryogenesis is the most feasible alternative to the other in vitro methods (Zimmerman, 1993, Saiprasad, 2001). Somatic embryos are widely considered to be of single cell origin; hence this is advantageous for transformation studies. Moreover, the process of somatic embryogenesis offers a mean to propagate large numbers of transgenic plants over a short period of time.

V. doniana seedlings (2-4 years old) originally wildings collected from the field in Glo in the south part and Cove in the central part of Benin were transported to Abidjan, Côte d’ Ivoire where they were maintained in a temporary shed and watered daily for one month before harvesting the leaves.

Explant preparation and surface sterilization

Healthy looking young leaves (2^nd pair) were collected and cleaned with cotton wool soaked with liquid soap. Thereafter, they were immersed in 0.5% (w/v) fungicide (Ridomil) containing two drops of Tween-20 (wetting agent) for an hour. The leaf explants were then transferred to the lamina flow cabinet for surface sterilization. Pre sterilization was carried using 70% ethanol solution for thirty seconds and then rinsing twice with sterile distilled water. The explants were subjected to further sterilization using varying (1, 1.5 and 2%) concentrations of calcium hypochlorite (CaOCl₂) containing 2-3 drops of Tween 20 and varying time duration. In an attempt to increase the number of clean explants, double sterilization (two steps) was conducted. This involved sterilizing the explants using 2% calcium hypochlorite for 30 min rinsing twice with sterile distilled water followed by quick dip in 70% ethanol. They were then subjected to a second step, by sterilizing them with 2% calcium hypochlorite for 15 min and finally rinsing with sterile distilled water four times.

Media preparation and culture conditions

Silver nitrate (at concentrations 8.45; 16.9 and 25.35 mg/l) and four amino acids namely proline (5.75; 11.5 and 17.25 mg/l), tryptophan (10.2; 20.4 and 30.6 mg/l), lysine (7.3; 14.6 and 21.9 mg/l) and leucine (6.55; 13.1 and 19.65 mg/l) were added to half strength Murashige and Skoog (MS) (1962) medium supplemented with 100 mg/l myo-inositol, 0.11 mg/l TDZ and 2% sucrose in separate experiments. The pH of the medium was adjusted between 5.7 and 5.8 prior to the addition of the solidifying agent, and autoclaved at 121°C for 15 min. The MS medium without AgNO₃ and amino acids is referred to as control. All cultures were incubated in a dark room maintained at 25 ± 2°C.

Surface sterilization

The highest  percentage  (70%)  of  clean  leaf   explants were obtained when 2% calcium hypochlorite was used for 20, 25 and 30 min, respectively (Table 1). However, this percentage decreased further and after two weeks only 50% clean explants were observed. The evaluation of double sterilization treatment resulted in increase in percent clean explants from 50 to 91% after one month and this procedure was used for all the subsequent experiments.

Effect of AgNO₃ on induction and regeneration of somatic embryos

The addition of AgNO₃ to the culture medium had a significant effect on the mean number of embryos per explant (Table 2). The percent embryogenic cultures decreased with increasing AgNO₃ concentration. The highest (91 and 90 %) frequency of embryogenic cultures were observed in the control and in the media supplemented with 8.45 mg/l AgNO₃. The later AgNO₃ concentration produced the highest (6.5) mean number of embryos. On the other hand, the lowest (1.2) mean number of somatic embryos was produced in the control. Significant differences were detected between the number of somatic embryos per explant at 8.45 mg/l AgNO₃ and the other concentrations of AgNO₃ at p < 0.01. Callus was observed from the cut edges of the leaf and globular embryos were obtained four weeks after culture (Figure 1A and B). Cotyledonary stage of embryos was observed two months after culture as shown in Figure 1C.

The current study was conducted with the aim of evaluating some of the factors that influence induction and regeneration of somatic embryos since there were no available reports on tissue culture of V. doniana. Before any explants is placed into culture, it is essential to destroy all microorganisms and the success in tissue culture depends on the effectiveness of the sterilization methods used on the explants prior to culture initiation (Yildiz and Celal, 2002). The outer surface of plants growing under natural or greenhouse conditions is normally infected with spores and other microbial cells. The use of field grown plants as a direct source of explant material for obtaining ‘clean’ explant, presents a major challenge. Previous attempts to initiate clean explants from field grown coffee, especially those from canopy close to the ground, resulted in 100% contamination (Kahia, 1999). In an attempt to obtain clean in vitro cultures, sources of contamination other than surface contaminants need to be considered. Even if the surface of the explant is effectively sterilized, the contaminants could emanate from the inner tissues when the plant materials are dissected into small explants after the surface sterilization. Systemic contaminants, for example, are not eliminated by surface sterilization (Webster et al., 2003). For this reason, a systemic fungicide such as Ridomil used in this study can be helpful to control the incidence of internal fungal infection in explants. The disinfectant widely used for surface sterilization of explants in tissue culture is sodium hypochlorite which dates back to the mid-18th century (Miche and Balandreau, 2001). It is usually purchased as household laundry bleach and as such it is readily available and can be diluted to proper concentrations. A balance between concentration and time must be determined empirically for each type of explant because of phytotoxicity. Previous reports indicate that bleach (1.4% NaOCl solution for 1 min) was found to be effective in sterilizing greenhouse-derived leaf explants in Aquilaria crasna and Aquilaria sinensis  (Okudera and Ito, 2009). On the other hand, effective sterilization was achieved by using 50% bleach for 20 min on shoot tips of Aquilaria hirta, from greenhouse (Hassan et al., 2011) while leaf explants of Allanblackia stuhlmannii were best surface sterilized using 8% sodium hypochlorite (Neondo et al., 2011). The other sterilant used for decontaminating explants is calcium hypochlorite. It is known to be less injurious to plant tissues and is generally used at a concentration of 3.25% (CSS 451 2009). During the present study, calcium hypochlorite was used as attempts to use the commercial bleach even at very low concentrations was found to be phytotoxic and led to the death of explants. The lower concentrations of calcium hypochlorite were not effective in decontaminating the explants. These results concur with those of Mihaljevi? et al. (2013) who reported high percent sterilization (80%) of sour cherry nodes when a high (3%) concentration of CaOCl₂ was used. A two-step sterilization was adopted in the work being reported in order to increase the percent clean explants and using this procedure, 91% clean explants was recorded. Similar results were reported by Nieves and Evalour (2011) who recorded 90% clean explants from cotyledon of Moringa oleifera using two steps comprising of 5% Ca(OCl)_2.