Seeds remain the most convenient and successful way for storing the genetic diversity of plant species and for producing new plants routinely for agriculture and horticulture. The importance of seed storage and the ability to predict seed longevity must therefore not be underestimated. To be successful, storage conditions must maintain seed vigour and viability and ensure that normal seedlings are subsequently established under field conditions. Seed quality is best retained when deteriorative events are minimised, which is achieved by storage of low moisture-content seeds under cool to cold, or even sub-zero, temperatures. Such conditions are employed for 'orthodox' seeds, which are desiccation tolerant and able to survive at sub-zero temperatures in the dehydrated state for extended periods. It is seeds referred to as 'recalcitrant' that cannot be dehydrated and often not stored at low temperatures because they are desiccation sensitive and may not tolerate chilling. According to almost anecdotal records chilling temperatures for such seeds are those below 15°C down to 0°C, depending on the species. The limited storage lifespan of recalcitrant seeds presents a problem even for short-term storage, and as most research on chilling sensitivity has been conducted on vegetative tissue, relatively little data exist for seeds, especially recalcitrant types. The purpose of this study was to gain an understanding of the chilling response of recalcitrant seeds, as reduced temperature could have the potential to extend, rather than curtail, storage lifespan, depending on the species. Selected physiological, biochemical and ultrastructural responses of recalcitrant seeds of Avicennia marina and Ekebergia capensis were characterised. Seeds of the two species were stored at 25, 16 and 6°C. Germination, water content (determined gravimetrically), respiration (measured as CO2 production) and leachate conductivity (tissue electrolyte leakage over time) were assessed at regular intervals. Chilling response at the subcellular level was examined using transmission electron microscopy (TEM). Changes in sugar metabolism and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were assessed for A. marina seeds, which were severely affected by the chilling temperature of 6°C, losing viability after 1 week. In contrast, the seeds of E. capensis retained viability after 12 weeks of storage at 6°C, indicating the marked difference in chilling response between seeds of the two recalcitrant species, despite their common tropical provenance. However, when E. capensis seeds were stored at 3°C viability decreased significantly after 8 weeks, thus indicating how critically temperature must be controlled if such conditions are to be profitably employed. Ultrastructural studies revealed that in both E. capensis and A. marina seeds vacuole formation was initiated more rapidly at lower temperatures than at higher temperatures, indicating that this was a response specific to the chilling stress imposed. Once again, 'lower temperatures' differed relative to the species concerned. In the E. capensis seeds, nucleolar morphology was affected and the extent of chromatin patches in the nuclei increased as the storage temperature was reduced. Other ultrastructural findings could not be linked specifically to the chilling stress imposed on the E. capensis and A. marina seeds. Activity of the antioxidant enzymes SOD and GR was detected in the A. marina seeds. No measurable CAT activity was detected. Glutathione reductase activity increased in response to chilling stress, the rate of the increase depending upon the severity of the chilling stress imposed. Other than when the A. marina seeds were placed directly at 6°C, there were no notable increases in SOD activity. Interestingly, SOD and GR activity was not the same in the axes as in the cotyledons. Superoxide dismutase activity was found to be higher in the axes and GR activity higher in the cotyledons. It would have been beneficial to determine the extent of antioxidant enzyme activity in the E. capensis seeds as well if this had been possible. Generally, chilling of recalcitrant seeds seems to evoke a response similar to that of dehydration below a critical water content. This could lead to the conclusion that recalcitrant seeds do not possess the genetic ability to cope with dehydration or chilling stress, if it were not for the existence of recalcitrant seed species that are more chilling tolerant. / Thesis (M.Sc.)-University of Natal, Durban, 2002.

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