We investigated the thermal thresholds to seed germination and the variations in abscisic acid (ABA) levels and oxidative metabolism during seed dormancy-breaking and germination in two palm species with differences in desiccation tolerance. We used Mauritia flexuosa (buriti palm, desiccation-sensitive seeds) from swampy habitats (Veredas) and Attalea speciosa (babassu, desiccation-tolerant seeds) from the transition zone between the forest and semi-arid region (drained soils). Germination was evaluated at 15–40°C after dormancy-breaking (operculum removal). At optimal temperature for both species (30°C), embryos were sampled in distinct germination phases – dry, imbibed, after operculum removal and at early germination – and used for quantifying ABA and hydrogen peroxide (H2O2) content, antioxidant enzyme activities and for histolocalization of superoxide anion (O2−). Seeds of M. flexuosa germinated only in a narrow temperature range (25–35°C), while A. speciosa seeds germinated between 15 and 40°C. After operculum removal, reduced ABA levels in embryos of M. flexuosa were accompanied by constant H2O2 levels, while in A. speciosa, similar levels of ABA and H2O2 were maintained throughout all germination phases. The presence of O2− was restricted to the haustorium, and an increase in O2− accumulation was observed in both species after operculum removal. Similarities were noted between both species regarding enzyme activities; however, the activities were higher in embryos from M. flexuosa. The presence of O2− only in the haustorium indicates that this region of the embryo is an active structure following imbibition and is involved in germination itself, not just functioning in reserve mobilization.

Inga vera subsp. affinis (Fabaceae) is a tree species native to riparian forests in Southeast Brazil and is key for the restoration of deforested areas. The species produce seeds that are highly recalcitrant. Extreme sensitivity to desiccation as well as vivipary are commonly observed in mature seeds, which also tend towards polyembryony. Past research has shown that typical strategies to store seeds are inapplicable to Inga vera as viability is completely lost when seeds are either dried to around 28% water content (wet basis) or stored at 5°C for a few weeks. Here, we examine the feasibility of storing the seeds under hydrated conditions but at reduced water potential. Freshly collected seeds were kept under conventional storage conditions (plastic bags in cold chamber, 5°C) and in polyethylene glycol (PEG) solutions (−1.6 and −2.4 MPa) at 10°C. Seed germination was assessed after various intervals of time, until all seeds had lost viability. Before storage, seeds attained 100% germination and produced an average of 1.8 normal seedlings per seed (due to polyembryony). Storage in PEG at −1.6 MPa maintained 90% germination (radicle protrusion) and one normal seedling per seed on average for more than 200 d. Osmotic storage likely slowed down metabolism within the seed and hence consumption of food reserves. The storage time achieved has practical applications for in situ restoration, but cannot address ex situ germplasm conservation. Extending shelf life for an additional 6 months allows tree nurseries to optimize the production of seedlings so that they can be planted during the wet season.

Changes in germination and desiccation sensitivity were measured throughout the seed expansion phase of development in fruits of Quercus robur L. The onset of a reduction in sensitivity to desiccation during development on the tree coincided with acquisition of the capacity for seed germination on moist sand substrate. Tolerance of desiccation then increased throughout development to shedding, but viability was still lost at a relatively high moisture content, and seeds did not therefore pass through a fully desiccation-tolerant phase. These data suggest that desiccation sensitivity in Q. robur may have resulted from the premature termination of development.

No desiccation was required to initiate germination in prematurely harvested fruits orseeds. Germination rate of seeds on moist sand increased at successive harvests during development, and was also increased by presoaking seeds in water. Variation in germination rate following shedding was not related to seed size or moisture content, but was affected by shedding date. This effect was not observed when the pericarp was removed.

The effects of temperature on dormancy loss, germination and viability were investigated in seeds of Aesculus hippocastanum L. harvested over a 4-year period. Release from embryo dormancy in freshly harvested seeds was manifest in two phases of morphological growth: initially, when the seed lot was only partially released, axis emergence resulted primarily from cotyledonary petiole extension without radicle extension; subsequently, when the seed lot was totally released, axis emergence of all seeds was followed immediately by extension to >1 cm through growth of the radicle. Germination (axis emergence and radicle extension) at 16°C was a function of pre-treatment period at 2–11°C. The rate of dormancy loss (probit germination d−1) increased linearly below a ceiling temperature for the chilling response; this temperature was estimated to vary from 13°C to 16°C for two seed lots harvested in separate years. Dormancy periods for individual seeds within both seed lot populations can be described by cumulative normal distributions; the predicted standard deviation of chilling units below the ceiling temperature (i.e. thermal time) was 186°C d. Visible germination occurred during the process of stratification at 2°C, starting after 21–25 weeks. By contrast, three years of hy-drated seed storage at 16°C, which was a non-permissive temperature for dormancy loss, resulted in little pre-emergence of the axis during stratification; approximately one third of the seeds remained germinable. The implications of these quantitative analyses of the physiological processes in recalcitrant seeds for the development of improved storage methods are discussed.

Embryos of Araucaria angustifolia seeds showed no dormancy; they germinated easily at temperatures ranging from 10° to 30°C, and the thermal optimum was about 25–30°C; they were recalcitrant. At harvest, their mean moisture content (dry weight basis) was about 120% and they completely lost viability when their moisture content fell to about 30%. The cotyledons were more sensitive to dehydration than the radicle. Dehydration induced deterioration of cell membranes as indicated by a high increase in leakage of solutes. It also resulted in damage in the nuclei, which was not repaired upon rehydration. During desiccation, respiratory activity decreased; however, O2 uptake was not an indication of germination ability, since it was significantly affected only when embryo moisture content reached the critical value of 30%. The decrease in the capacity to convert 1-aminocyclopropane 1-carboxylic acid to ethylene, which was observed at 30–60 min of dehydration, was a very early indicator of deterioration in embryos. Desiccation resulted also in a rapid decrease in the ability for protein synthesis as measured by [35S]methionine incorporation into total protein; 50% inhibition was observed after 30–60 min of desiccation for both axis and cotyledons.

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.

Abstract An autoradiographic study was made of leucine and thymidine incorporation into the meristematic root primordia and hypocotyl tips of seeds of the recalcitrant mangrove species, Avicennia marina. The investigations show that although there is a temporary reduction of protein synthesis at shedding, root primordia and surrounding hypocotyl cells of the axis never wholly cease incorporation of [3H]leucine and regain pre-shedding levels of activity within a day. Precursor studies using methyl-[3H]thymidine show that, at shedding, there is a temporary cessation of incorporation into root meristem nuclei that lasts no longer than 48 h and, within a day, pre-shedding levels are regained in the meristem nuclei. Analysis of DNA fragmentation patterns in root tips at the time of shedding, and their ability to repair radiation-induced DNA damage, indicate that DNA repair processes are markedly compromised in these cells if water loss reaches 22%. Protein synthesis and DNA replication are reduced by more than half by a water loss of 18% and 16%, respectively. DNA replication does not fully recover on rehydration after only 8% water loss. DNA fragmentation to nucleosomes indicates a programme of cell death at a water loss of 10%. We suggest that the feature of continuous protein synthesis activity with only a temporary interruption in active cell cycling in A. marina root primordia helps to explain both the rapidity in seedling establishment and the extreme vulnerability to desiccation.

The aim of the present study was to examine the micromorphology of the cork-oak (Quercus suber) acorn and to evaluate the efficiency of the pericarp as a barrier to water loss. When the desiccation curve obtained at 25 ± 1°C and 62% RH for physiologically mature acorns collected from the tree was compared with that corresponding to fully ripe acorns which had been shed, the latter showed a slightly lower rate of water loss. A marked reduction in the mean rate of water loss was recorded for intact acorns compared with those from which the pericarp had been removed. The external surface and transverse sections of the pericarp from both mature and fully ripe acorns were examined by scanning electron microscopy. Two zones presenting morphological and micromorphological differences were identified: an area including the point of attachment to the cupule and an apical zone covering the embryo. The microstructure of the pericarp showed an external thick cuticle and a single external palisade layer of closely packed cells with no intercellular spaces. Underlying this layer, there was a further parenchymatous layer of poorly differentiated, roughly isodiametric cells. The pericarp in the cupular zone consisted of only this undifferentiated layer between the two epidermal cell layers and contained vascular bundles with many xylem elements.

The desiccation sensitivity of seeds of Inga vera Willd. subsp. affinis, a recalcitrant-seeded tree from Brazil, was analysed, focusing on water relations and cell-cycle aspects, including DNA content and the microtubular cytoskeleton. Seeds were collected at four developmental stages, dried to different moisture contents (MCs), assessed regarding water activity and set to germinate. Samples of fresh (non-dried) developing and mature seeds were used for assessment of DNA content by flow cytometry. Immunohistochemical detection of microtubules (MTs) was done in mature seeds at different MCs. Slight desiccation of immature seeds increased germination, but further drying resulted in a quick decline of germinability. During seed development the desiccation sensitivity decreased slightly, but DNA content of the embryonic axis cells remained constant, suggesting no relation between those two parameters. Embryonic axis cells of mature seeds showed abundant cortical microtubule arrays, which were not affected by mild desiccation, but broken down by further drying. It appeared that, upon rehydration, damaged cells were not able to reconstitute the microtubular cytoskeleton. The failure of germination of Inga vera seeds after drying could not be attributed to cellular damage to DNA synthesis and mitosis, since the radicle protruded by means of cell elongation, without a need for cell division. However, the breakdown of MTs during desiccation, and their subsequent inability to reassemble upon rehydration, may be related to the decreased germination, since MTs are required for cell elongation.

This study examined the hypothesis that desiccation sensitivities of recalcitrant and intermediate seeds can be categorized into discrete levels of critical water potential. The equilibrium dehydration method was used to determine the critical water potential (CWP) below which desiccation damage started to occur. The CWP values of Bruguiera cylindrica, Lansium domesticum, Litchi chinensis and Lumitzera racemosa are approximately –4 MPa. The CWP values of Andira inermis, Avicennia alba, Castanea sinensis (from New Zealand), Citrus aurantifolia, Ginkgo biloba, Nephelium lappaceum and Theobroma cacao (immature axis) are approximately –8 MPa. The CWP values of Acer pseudoplatanus, Castanea sinensis (from China), Quercus rubra and Theobroma cacao(mature axis) are approximately –12 MPa. The CWP values of Artocarpus heterophyllus and Hevea brasiliensis are approximately -23 MPa, while the CWP values of Acer platanoides, Azadirachta indica, Carica papaya and Coffea arabica are approximately –73 MPa. Together with data available in earlier literature, these CWP values suggest that there are five discrete levels of critical water potential among desiccation-sensitive seed tissues. These data support the hypothesis that discrete levels of desiccation sensitivity occur among recalcitrant and intermediate seeds, and suggest that specific damaging and protective mechanisms exist at certain hydration levels.

The decline of embryo moisture content from approx. 82 to 53% in 1997 and 56% in 1998 in recalcitrant seeds of Acer saccharinum during maturation was accompanied by decreased mitotic activity in the meristems and an increase in the percentage of cells in the G1 phase of the cell cycle. DNA synthesis and mitosis in the root apex ceased at approx. 53% embryo moisture content, and 67% of the cells were arrested in the G1 phase. During post-maturation drying, cell division in the shoot apex and embryonic leaves continued as long as the embryo moisture content was higher than 50 and 45%, respectively. Mitotic activity in the drying embryo may be controlled by its moisture level. Increased proliferation of the root, shoot and leaf meristems of the mature seeds was already recorded at 24 h of germination, before the root protruded through the seed coat. However, the increase in the embryo moisture content was small – from 56 to 59%. In the 3 d seedlings (10–15 mm long) the mitotic index reached 8% in the root apex and 12% in the shoot apex with leaves. Placing mature seeds in moist conditions might be necessary for the switch from proliferation decline towards its activation. Thus, in contrast with orthodox seeds, the preservation of cell division capacity and increased mitotic activity may be essential for rapid germination immediately after shedding in mature Acer seeds.

The plumules of newly-excised wampee embryos, which are more sensitive to dehydration than the roots, became more resistant to water loss when axes were allowed to sprout on woody plant medium [WPM; McCown and Lloyd (1981) Hortscience16, 453] before being dried. Pre-treatment of sprouting axes (seedlings) with sucrose incorporated in the WPM enhanced survival. Although the roots withered following further dehydration of seedlings cultured on WPM containing 60% sucrose, excised plumules were capable of generating adventitious roots when a combination of 10 mM α-napthaleneacetic acid and 10 mM indole-3-butyric acid was used during subsequent in vitro incubation.

Mature and immature axes of Theobroma cacao (cocoa) seeds tolerated desiccation under a rapid-drying regime to critical water contents of 1.0 and 1.7 g g-1 dw, respectively. These critical water contents corresponded to water contents below which activities of free radical-scavenging enzymes (ascorbate peroxidase, peroxidase and superoxide dismutase) decreased rapidly during desiccation. The decline in axis viability below the critical water content was correlated with sharp increases in lipid peroxidation and cellular leakage. Cotyledon tissues were more desiccation-tolerant than axes, with a low critical water content of 0.24 g g–1dw. Desiccation sensitivity in cotyledon tissues was also correlated with the decrease in superoxide dismutase activity and increased lipid peroxidation products. However, in the cotyledons, no ascorbate peroxidase activity was detected at any water content, and peroxidase activity was gradually reduced as desiccation proceeded. Cocoa embryonic axes contained large amounts of sucrose, raffinose and stachyose but only traces of reducing monosaccharides. Desiccation sensitivity of recalcitrant cocoa axes did not appear to be due to the lack of sugar-related protective mechanisms during desiccation, and it was more likely related to the decrease of enzymic protection against desiccation-induced oxidative stresses.