LAUSR

Somatic embryogenesis is an ideal technique for the micropropagation of date palm using different explant tissue; however, histological studies describing the ontogenesis of plant regeneration are limited. This chapter provides a simple protocol for the histological analysis of the successive developmental stages of direct somatic embryogenesis induced from in vitro leaf explants. Direct somatic embryos are obtained from Murashige and Skoog (MS) medium containing 2 mg/L 6-benzylaminopurine. In order to observe the different developmental stages, histological analysis is carried out on samples at 15-day intervals for 60 days. Samples are fixed in formalin acetic alcohol and embedded in paraffin wax. Stain serial transverse and longitudinal sections, 8 μm thick, are stained with safranin-Fast Green. After 15 days on the induction medium, somatic embryos exhibit multicellular origin directly from the procambium cells, whereas the mesophyll and the epidermal cells are not involved in this process. After 2 months, several developmental stages (pre-globular, globular, early bipolar, bipolar, and cotyledonary-shaped) are observed. These embryos germinate after transferring to MS medium without plant growth regulators and rooting on 2 mg/L NAA-containing medium resulting in complete plantlets.