A simplified Protocol to Induce Callogenesis in Protoplasts of Date Palm (Phoenix dactylifera L.) Cultivars

Background: In Algeria, date palm is currently confronted to the Bayoud disease. Biotechnological tools such as protoplasts fusion can appear as an alternative to ensure rapid multiplication and improvement of this species. Objectives: Callogenesis induction in protoplasts isolated from embryogenic callus of three date palm cultivars. Materials and Methods: Some factors influencing the isolation and culture of protoplasts segregated from the calli of three date palm (Phoenix dactylifera L.) cultivars (Deglet Nour, Akerbouch and Degla Beida) were studied. Protoplasts of each cultivar were cultured on a semi-solid medium supplemented with various hormonal balances. Results: Maceration with an enzymatic solution containing 1.5% cellulase and 1% macerozyme R10 in the presence of 0.5 M mannitol for more than 16 h with gentle agitation allows isolation of a great number of viable protoplasts. In addition, purification of protoplasts on a cushion of 21 or 25% sucrose was effective in cell debris removal and maximum recovery. The culture of isolated protoplasts on a semi-solidified Murashige and Skoog medium, with 0.3% agarose, 2 mg. L-1 2,4-D and 0.5 mg.L-1 BAP allowed good viable protoplast maintenance as well as cell wall regeneration. After more than two months of culture, cell divisions were still occurring and microcalli became visible to the naked eye, containing a large number of cells. Conclusions: The developed protocol can be useful for application of somatic hybridization to improve date palm cultivars.