A novel means for cryopreservation of germplasm of the recalcitrant-seeded species, Ekebergia capensis.

Cryopreserved zygotic embryonic axes offer the best means of genetic diversity conservation of recalcitrant-seeded species, but frequently shoots fail to develop following processing for, and after, cryostorage. The present work offers a means to overcome this, by generating adventitious shoots from seedling roots produced after axis cryopreservation. Embryonic axes of Ekebergia capensis were exposed to cryoprotectants, flash dried, and rapidly cooled in nitrogen slush. Cryoprotection was an essential step, with both glycerol and DMSO permitting survival after cryogen exposure, but sucrose alone, or in combination with glycerol, was deleterious. Adventitious shoots were formed from seedling roots developed by axes germinated after cryogen exposure, after being subjected to intermittent flushing with a BAP-containing medium for 24 h in a temporary immersion system and subsequent culture on a semi-solid BAP-containing medium. After excision, a high proportion of the adventitious shoots produced roots in vitro, with most of these rooted plantlets being subsequently successfully acclimated.