Cryopreservation of apple shoot tips by encapsulation-dehydration: Effect of preculture, dehydration and freezing procedure on shoot regeneration

Shoot tips sampled on in vitro cultured apple plantlets of 6 accessions of M. domestica and one accession of M. robusta were successfully cryopreserved using the encapsulation-dehydration technique. Shoot tips were excised from plantlets which had been submitted to 3 weeks of cold-acclimation at 5 degrees C, 70 d after their last subculture. After preculture at 5 degrees C in media with progressively increased sucrose concentration (0.1 M,0.3 M and 0.7 M), shoot tips were encapsulated and pregrown in medium with 1.0 M sucrose for I d, dehydrated for 4 h under the air current of the laminar flow cabinet, thus reaching a moisture content of around 30% (fresh weight basis) and directly immersed in liquid nitrogen. The regeneration rate of cryopreserved apices varied between 70 and 90%, depending on the accession. Using apices sampled on plantlets which had been maintained on standard medium without subculture for 6 months, sucrose preculture became unnecessary to achieve regrowth after cryopreservation and the dehydration period was shortened. These experiments showed that the physiological state of the plant material directly affects the results and procedures for cryopreservation of apple shoot tips.