Cryopreservation of date palm (Phoenix dactylifera L.) cultured in vitro

A very simple cryopreservation method with potential application to a wide range of date palm cultivars is described. Undifferentiated tissue cultures were successfully cryopreserved by freezing methods and plants were subsequently regenerated. Nodular cultures were initiated by culturing shoot tip explants (excised from offshoots) on medium containing 10 mg/L dichlorophenoxyacetic acid (2,4-D) + 3 mg/L dimethylaminopurine (2ip). The effect of dehydration caused by air drying on cryopreservation of date palm tissue cultures through direct immersion in liquid nitrogen was investigated. Cultures with about 65% water content resulted from a 20 min air drying period registered the highest percentage of survival and in vitro conversion to plantlets. Of the different types of sugars used as osmotic agents in a preculture medium, sucrose was the best for the survival of cryopreserved date palm tissue cultures. To determine the effect of vitrification on freezing tolerance, cultures were exposed to a vitrification solution (22% glycerol, 15 % ethylene glycol, 15% propylene glycol, 7% dimethyl sulfoxide) at 25 or 0 degrees C for 20-100 min. The maximum rate of survival was obtained with cultures exposed for 80 min at 0 degrees C followed by 40 min at 25 degrees C. Random Amplified Polymorphic DNA (RAPD) technique was used to study the genetic stability of cryopreserved tissue cultures of date palm. According to RAPD analysis, plantlets derived from cryopreserved cultures were identical to those derived from nontreated cultures and both were similar to field grown plants. Finally, complete plantlets retrieved from the cryostored cultures were successfully adapted to free living conditions after acclimatization procedures.