As a consequence of previous lack of success in cryostoring axes excised from newly shed seeds of Trichilia dregeana, the effects of the mode of axis excision on seedling production were investigated. Although vigorous root production occurred, no shoots were produced when the cotyledons were severed as closely as possible to the axis surface (explant-type 0). In contrast, shoot production was increasingly facilitated when small to larger segments of cotyledonary tissue were left attached to the axes (explant-types 1, 2 and 4), which did not compromise axis drying rate. However, root growth of explant-types 1, 2 and 4 was negatively affected, probably by leakage into the medium of an inhibitory or toxic substance(s) from the cut surfaces of the cotyledonary tissue. Microscopical examination revealed that the cotyledons were sessile, and their insertions were contiguous with the shoot apex in axes from newly shed seeds, leading to the suggestion that failure of shoot production by type 0 explants in vitro was the direct consequence of the proximity of the wound sites to the apical meristem. When seeds were stored hydrated for 6 months, the shoot apex had elongated, positioning the apical meristem some distance from the top of the cotyledonary insertions. In contrast to axes excised as type 0 explants from newly shed seeds, the equivalent explants from the stored seeds rapidly formed shoots and leaves in vitro. This indicates that the developmental status of axes, when excised, dictates failure or success in their further development in vitro, and that this aspect needs to be resolved before any further manipulations for cryostorage are attempted.

Assessment of the rehydration procedure has been consistently overlooked in evaluation of factors contributing to successful cryostorage of partially dehydrated, embryonic axes excised from recalcitrant seeds. Conventional rehydration of Trichilia dregeana (Sond.) axes in distilled water resulted in the lack of root pole gravitropism after culture on medium in vitro. In comparison, a strong gravitropic response was observed in axes that had not been dehydrated, and by those rehydrated in a solution containing 1 M CaCl2 and 1 mM MgCl2. However, no marked loss of either cation from the tissue could be detected, whether axes were rehydrated in distilled water or the Ca2+/Mg2+ solution. Starch-packed statoliths differentiated rapidly in both non-dehydrated axes and those rehydrated in the divalent cation solution, but these organelles failed to develop or accumulate much starch following axis rehydration in water, as was the case for the amyloplasts of root cells generally. After rehydration in the Ca2+/Mg2+ solution and 48 h in culture, axis root-cap columella cells accumulated Ca2+, whereas axes rehydrated in water or solutions containing either Ca2+ or Mg2+ alone did not take up Ca2+. Rehydration with the Ca2+/Mg2+ solution also altered distribution of the actin component of the cytoskeleton. When rehydrated in the divalent cation solution, actin was associated with the nucleus and with the statoliths, which were located distally in statocytes of axes. In contrast, actin was largely confined to the perinuclear area in root-cap columella cells of the agravitropic, water-rehydrated axes. The present results indicate a definitive primary role for Ca2+ with Mg2+ in graviperception, via starch metabolism, and in the determination of statolith morphology, which appears to be linked with maintenance of the actin component of the cytoskeleton in root-cap statocytes.

Recalcitrant seeds of Trichilia dregeana were stored at 16 or 25°C, either at the water content at which they were shed or partially dried. Although having been exposed to a short period (approx. 6 h) at temperatures up to 30°C prior to storage, seeds at the original water content maintained viability for several weeks at 16°C. However, storage of undried seeds at 25°C was deleterious within 8 d, indicating a chemical basis for degeneration of hydrated recalcitrant seeds. Seeds that had been mildly dehydrated to the relatively high axis level of 1.68 g H2O g—1 dry mass, while maintaining full germinability immediately after drying, exhibited only 4% viability after 8 d in storage at 16°C and had completely lost viability after the same storage period at 25°C. Ultrastructural features characterizing hydrated seeds included indications of enhanced activity associated with initial exposure to the elevated temperature as well as some signs of stress. However, over an effective 15 d storage period at 16°C, ultrastructural features showed the cells to have retained little damage and to have been in an enhanced state of activity commensurate with ongoing development towards germination. After a longer storage period, however, signs of damage, including indirect evidence for disarray of the cytoskeleton in some axis cells, became apparent in line with the declining seed viability. Immediately following dehydration from an average axis water content of 1.97 to 1.68 H2O g g—1(the sub-imbibed condition), some ultrastructural abnormalities were apparent, but the seeds remained 100% germinable. However, within the 8 d storage period in this sub-imbibed condition, a spectrum of severe ultrastructural degeneration, including indirect evidence of the collapse of the nucleoskeleton and extensive cell lysis, accompanied viability decline of the seeds to 4%.