A study of some chilling responses of recalcitrant seeds of Avicennia marina (Forssk.) Vierh. and Ekebergia capensis Sparrm.

Abstract

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.