Responses to chilling and cryopreservation of recalcitrant seeds of Ekebergia capensis from different provenances.

Abstract

Recalcitrant seeds are shed at relatively high water contents and are metabolically active. The effect of chilling the recalcitrant seeds of purportedly a single species, Ekebergia capensis, from Port Elizabeth (PE; Eastern Cape), St Lucia (KwaZulu-Natal [KZN]) and Tanzania (tropical southern Africa) was tested. Viability and axis ultrastructure, solute leakage and protein synthesis were investigated. Additionally, cryopreservation of embryonic axes (explants) was studied. In particular, the ability of cathodic water to improve explant survival was tested, and related to its effect on the production of potential harmful reactive oxygen species such as superoxide (·O2-) and hydrogen peroxide (H2O2), and its ability to maintain levels of total aqueous antioxidants (TAA). Shoot and root ultrastructure were examined after each cryopreparative step in explants treated with and without cathodic water, and ultrastructure correlated with survival. The seeds from PE retained 80% viability after 12 weeks storage at 1° and 3°C and 100% when stored at 6°C. Those from St Lucia were dead after 38 d storage at 3°C. All the seeds from Tanzania were dead after 9 d when stored at 3°C. The rate of protein synthesis decreased gradually over the storage period, irrespective of the provenance. Electrolyte leakage from axes showed that those from St Lucia and Tanzania ‘leaked’ solutes irrespective of the duration or temperature or storage, but those from PE showed an initial increase, which then decreased. A 30% mass loss was achieved after 48 d, 230 h and 21 h for seeds from PE, St Lucia and Tanzania, respectively. Nuclear ribosomal ITS1 sequences revealed the presence of three well-to-strongly-supported monophyletic clades corresponding to the geographical areas from which the seeds were sampled (PE, KZN and Tanzania). Axes from the seeds from St Lucia and Tanzania lost ultrastructural integrity during storage while those from PE did not. The levels of ·O2- and H2O2 increased gradually after each cryopreparative step. Using cathodic water allowed 30% of PE explants to survive cryopreservation, while none of the St Lucia explants did. Root ultrastructure was well preserved, however, gradual ultrastructural deterioration was observed in the shoot meristem.