Cryopreservation of pineapple (Ananas comosus (L.) Merr) apices and calluses

Cryopreservation of apices is the only option for long-term conservation of pineapple germplasm. It is also of high interest for this crop in the framework of biotechnology programs for conserving calluses, which are employed for in vitro resistance assays to fusariose and transformation works. This current study was aimed at developing cyropreservation protocols for pineapple apices and calluses. The vitrification technique was used with apices sampled from in vitro and a simplified freezing process was used with calluses. With apices, optimal conditions included a 2-d pre-culture on a solid medium supplemented with 0.3 M sucrose, loaded for 25 min at 25 degrees C in liquid medium with 0.4 sucrose + 2M glycerol, treatment with PVS3 vitrification solution at 0 degrees C for 7h before immersion in liquid nitrogen. This method achieved 45, 33 and 25% survival with varieties Smooth Cayenne, Cabezona and Red Spanish, respectively. Fully developed plantlets could be obtained from cryopreserved apices. Calluses were pretreated with a cryoprotective solution containing 15% DMSO and 0.5M sucrose for 1h at 0 degrees C. Cryotubes were transferred to an alcohol bath which was placed in a -40 degrees C freezer, thus allowing a freezing rate of 0.4-0.6 degrees C/min between 0 degrees C and -40 degrees C, then immersed in liquid nitrogen. This method resulted in 57% and 65% survival for calluses of varieties Smooth Cayenne and Perolera, respectively.