Date palm (Phoenix dactylifera L.) cv. Khenizi caulogenic meristems were initiated from achlorophyllous leaves excised from in vitro shoot cultures and then proliferated on a specific culture medium supplemented with 70 g dm-3 sucrose. Regeneration rates obtained when using standard vitrification, droplet-vitrification, and encapsulationvitrification protocols reached 26.7, 60.0, and 40.0 %, respectively. Only explants smaller than 3 mm in diameter were found to survive cryogenic treatments. Sucrose preculture, cold hardening and loading solution pretreatments showed significant effects on regeneration rates. Moreover, our results indicate that both sucrose preculture and cold acclimation of explants increased proline content. Cryopreservation of date palm tissue with high proliferation capacity can directly benefit large scale micropropagation projects. (Résumé d'auteur

This chapter describes an efficient protocol for large-scale micropropagation of date palm. Somatic embryo-derived plants are regenerated from highly proliferating suspension cultures. Friable embryogenic callus is initiated from juvenile leaves using slightly modified Murashige and Skoog (MS) medium supplemented with 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures consisting of proembryonic masses are established from highly competent callus for somatic embryogenesis using half-strength MS medium enriched with 0.1 mg/L 2,4-D and 300 mg/L activated charcoal. The productivity of cultures increased 20-fold when embryogenic cell suspensions were used instead of standard protocols on solidified media. The overall production of somatic embryos mostly exceeds 10,000 units per liter per month. Partial desiccation of mature somatic embryos, corresponding to a decrease in water content from 90 down to 75%, significantly improved germination rates.

Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation. In this chapter, we describe three cryopreservation protocols (standard vitrification, droplet vitrification, and encapsulation vitrification) for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium. The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. Regeneration rates obtained with standard vitrification, encapsulationvitrification, and droplet-vitrification protocols can reach 30, 40, and 70%, respectively. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials. When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.