Cryopreservation of Date Palm Pro-Embryonic Masses Using the D Cryo-plate Technique.

Abstract

In this chapter, we describe a cryopreservation (liquid nitrogen, -196°C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171g/L sucrose for 3days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20min with a loading solution containing 184g/L glycerol and 136.8g/L sucrose. They were then dehydrated for 90-120min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15min in an unloading solution containing 410.4g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3days in the dark on posttreatment medium containing 102.6g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.