The challenge of successful cryopreservation of olive (Olea europaea L.) shoot tips

In vitro approaches, including cryopreservation, may provide an important component for a sustainable olive (Olea europaea L.) germplasm conservation strategy. Although there are reports of the successful recovery of olive shoot tips aftercryopreservation to date none present evidence of sustained post-thaw regrowth. In this study, the effectiveness of several cryopreservation approaches was compared. No post-thaw shoot tip regrowth was observed after either encapsulation/dehydration or encapsulation-osmoprotection/dehydration protocols. Post-thaw regrowth of 'Frantoio' shoot tips (up to 38%) was achieved following a "two-stage" incubation with PVS2 (50% PVS2 for 30 min, plus 100% PVS2 for 1 hr), direct plunging into liquid nitrogen and post-thaw culture on OM medium containing a high concentration (46 ?M) of zeatin. Despite the modification of zeatin and gibberellic acid combinations in the post-thaw culture medium, long-term regrowth of shoot tips could not be sustained significantly beyond 10 weeks. Histological examination indicated that the PVS2 treatment and subsequent plunging into liquid nitrogen of shoot tips did not modify the structure of the meristematic apex; however, several changes were noted in the sub-apical cells following PVS2 treatment, such as cell wall gelification of sub-apical cells, significant dehydration of external parenchyma cells and cellular starch accumulation. These changes became more enhanced by immersion in liquid nitrogen. The significance of these results in terms of olive cryopreservation is discussed.