Responses to cryopreservation of recalcitrant seeds of Ekebergia capensis from different provenances

Abstract

Recalcitrant seeds are shed at relatively high water contents and are metabolically active, initiating germination without exogenous provision of water. There is an urgency for these seeds to be conserved in light of the rapidly diminishing numbers of indigenous species and associated genetic diversity. This may be achieved by long term storage of whole seeds or seed germplasm. This study centred on the development of a cryopreservation protocol for embryonic axes (explants). Indicators of stress i.e. superoxide (·O2−), hydrogen peroxide (H2O2) and total aqueous antioxidants (TAA) levels were measured, as well as explant viability and shoot and root ultrastructure in response to exogenously applied cathodic water after each cryopreparative step.Explant viability gradually decreased after each cryopreparative step indicating the cumulative effect of the stresses imposed by cryopreservation. In conjunction with reduced viability, the levels of ·O2− and H2O2 increased gradually after each of these steps. Total aqueous antioxidant levels remained relatively unchanged throughout the cryopreservation process. The ROS levels in cathodic water treated explants were significantly lower compared with those that were not treated with cathodic water. A small number of explants from seeds obtained in Port Elizabeth (30%) survived after cryopreservation, while none of the explants from seeds obtained in St Lucia did.Root ultrastructure across explants from seeds of both provenances was well preserved after each cryopreparative step. Organelles were well developed and the ultrastructure was typical of metabolically active tissue. However, gradual ultrastructural deterioration was observed in the shoot meristem after each cryopreparative step. Typical of a stress response, increased vacuolation and a lack of visible organelles were noted. Root and shoot production (or lack thereof) was supported by ultrastructural observations.This study reports that the explants of PE origin were able to survive cryopreservation while those of St Lucia did not. The key findings of this study support the role of cathodic water in regulating ROS and indirectly preserving explant ultrastructure and survival, and its exogenous application during cryopreservation of recalcitrant seeded germplasm is recommended.