Some investigations of the responses of Quercus robur and Ekebergia capensis embryonic axes to dehydration and cryopreservation.

Thesis (M.Sc.)-University of Natal, Durban, 2000.

Recalcitrant seeds are those that are shed at high water contents, are actively metabolic throughout development, when they are, and remain, desiccation-sensitive, and may also be chilling sensitive. These properties preclude their conventional storage. Because recalcitrant seeds lose viability rapidly (within a few days to several months depending on the species) the long-term storage of their germplasm is achievable only by cryopreservation [i.e. storage at very low temperatures, generally in or over, liquid nitrogen at -196°C or -150°C, respectively. Generally the seeds are far too large to be cryostored, thus explants - most conveniently, excised zygotic embryonic axes - are used. As the axes of recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing have to be applied in order to avoid lethal ice crystal formation. During the course of this study, cryopreservation protocols were developed for excised zygotic embryonic axes of two different species (Quercus robur L. and Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes of both species, with the use of a 1% sodium hypochlorite solution containing a wetting agent, emerging as the best. For both species, the vigour and viability of axes, assessed by in vitro germination performance, was tested after the implementation of four different rates of desiccation (achieved by a laminar-airflow; silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate (fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur and E. capensis axes after 240 and 20 min, when water contents were reduced to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively. Consequently, fast flash-drying was used in combination with three different freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was lost after slow or intermediate cooling, good survival was obtained for each species after ultra-rapid cooling. In addition to the optimisation of culture conditions, desiccation and freezing rates, the efficacy of different thawing media (distilled water, mannitol, sucrose, full-strength MS medium supplemented with sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The only thawing medium that ensured normal seedling production was the Ca2+Mg2+ solution, in which electrolyte leakage was significantly curtailed. In addition to vigour and viability assessment the responses of the embryos to the various manipulations were monitored by light microscopy and/or transmission electron microscopy. The results of the various manipulations are discussed in terms of the stresses imposed on the excised axes, by each of the procedures. For axes of Q. robu, the outcome of the presently developed successful procedure and two unsuccessful protocols from the published literature are compared and contrasted. It is concluded that while in vitro germination media need to be assessed on a species basis, use of the mildest effective surface-sterilant, in conjunction with the most rapid means to achieve dehydration and cooling/freezing, are likely to underlie generally successful cryopreservation. Additionally, thawing parameters have emerged as being critically important.