Callogenesis induction on different types of explants of Diospyros crassiflora Hiern

The jet-black to streaked ebony wood produced by the African ebony (Diospyros crassiflora Hiern, Ebenaceae) is exploited in Central and West Africa. A conservation effort is currently underway in Cameroon to propagate the tree through seedlings and cuttings. However, the intermittent availability of its seeds and the long time required for rooting formation of cuttings are limiting its propagation. The aim of this research was to establish a reproductible protocol of the in vitro callogenesis induction in D. crassiflora. The young leaves below the apical meristem are taken from seedlings about 3 months old and derived from the germination of D. crassiflora seeds. Similarly, immature flower buds are taken from mature trees. These various explants are washed under a stream of tap water for 30 min and sterilized according to well-defined protocols . Leaf fragments were placed 15 explants per petri dish and repeated 4 times. For the staminodes, we grew 50 explants per petri dish and the experiment was repeated 3 times. For cotyledons, the mature seeds of D. crassiflora, once removed from the fruit, were thoroughly washed with tap water and then sanitized. The extraction of the cotyledons from its housing was done with a scalpel with a size 11 scalpel blade. The cotyledon fragments were placed 10 explants per Petri dish due to 6 repetitions. The experimentation lasted 28 days for each treatment for the leaves and cotyledons finally to observe the calluses. While in staminodes 133 days were necessary to observe callus induction.