How some plant cells can survive a period of desiccation to live again when other cells will die, was the theme of the recent Workshop in the mountain retreat of Franschhoek, near Cape Town, South Africa. Sixty-two biochemists and cell physiologists from around the world assembled to discuss these problems for the second time in three years.

Soluble carbohydrates are one of multiple components required for the acquisition of desiccation tolerance during seed development and maturation. Sucrose and the raffinose series of oligosaccharides have been extensively studied in relation to seed desiccation tolerance. These galactosyl sucrose oligosaccharides are present in viable tissues of many edible seeds, especially the legumes, and contribute to flatulence after ingestion. A reduction in oligosaccharides of the raffinose series is desired by nutritionists but, if present at less than a threshold level, this this may result in reduced desiccation tolerance and storability of seeds. Some seeds that have very low amounts of raffinose and stachyose accumulate galactosyl cyclitols and small amounts of free cyclitols. Galactosyl cyclitols present in various seeds include galactosyl or methyl derivatives of myo-inositol, D-pinitol, D-chiro-inositol, D-ononitol, and scyllo-inositol. Castor bean seeds accumulate galactinol and buck-wheat seeds accumulate fagopyritol in addition to sucrose. Fagopyritol accumulation is associated with the acquisition of desiccation tolerance in buckwheat seeds. The galactosyl sucrose and galactosyl cyclitol soluble carbohydrates in maturing seeds have been proposed to have various roles in desiccation tolerance including non-toxic and non-reducing forms of seed storage products and intracellular osmotic agents contributing to the structural stability of organelles, membranes, enzymes and proteins, other macromolecules, and the glassy state. Ether derivatives of cyclitols may form liquid crystals. Methyl ether derivatives of cyclitols may have roles as cryoprotectants, desiccation protectants, and hydroxyl radical scavengers.

Plants undergo a series of physiological, biochemical and molecular changes in response to adverse environmental conditions or stresses such as drought, low temperature or high salt. Several genes and their corresponding proteins have been described that may play a role in withstanding water-deficit-related stresses or full desiccation. In particular, sugars and late-embryogenesis-abundant (LEA) proteins have received the most attention. Plant responses to water-deficit and desiccation have been well-characterized at the molecular level; however, pinpointing the precise roles of the gene products in protecting cells under conditions of water deficit remains a challenging task. While few plants are capable of withstanding full desiccation, most seeds undergo this event as a pre-programmed and final stage in their development. These are the so-called ‘orthodox’ seeds. In contrast to seeds of orthodox species, those of recalcitrant species do not acquire desiccation tolerance during their development and are shed from the parent plant at relatively high water contents. The essential components of desiccation tolerance of seeds are likely to involve the ability to effect repair upon subsequent rehydration as well as the ability to accumulate protective substances that limit the amount of damage which otherwise would be caused by water loss. Studies have begun to examine whether the desiccation sensitivity of recalcitrant seeds is at least partially the result of an insufficient accumulation of LEA-type proteins, or whether other factors (including a lack of protective sugars) are more important. This review assesses some of these studies as well as recent research to understand gene and protein function using transgenic host plant systems.

It is proposed that desiccation tolerance in the embryo of seeds depends upon the capacity to repair damage to genomic DNA when the desiccated embryo is rehydrated. From a study of imbibed and hydrated embryos of rye (Secale cereale) and wild oat (Avena fatua) evidence is provided that it is neither the extent of water uptake by the cells, the ensuing stability of the DNA to desiccation, nor the onset of S-phase DNA synthesis in the first cell cycle of germination that determines whether the desiccated embryo will survive. It is shown that when α- and β-polymerases of DNA repair are inhibited by aphidicolin and dideoxythymidine-5'-triphosphate, respectively, a γ-irradiation-induced DNA fragmentation cannot be fully repaired. It is shown that in hydrated embryos, at a stage when desiccation tolerance is lost, embryo cells still repair irradiation-induced damage, but this repaired DNA is unstable to desiccation and cannot be rerepaired when water is again made available. The failure to re-repair on rehydration appears to be critical to embryo survival and successful germination.

Maturing yellow lupin seeds were desiccation tolerant. Glucose, sucrose and cyclitols (mainly D-pinitol, D-chiro-inositol and myo-inositol) were predominant at the early stages of seed growth. Accumulation of the raffinose family oligosaccharides (RFOs) and the galactosyl cyclitols including galactinol, digalactosyl myo-inositol, galactopinitol A, galactopinitol B, trigalactopinitol A, ciceritol, fagopyritol B1 and fagopyritol B2 appeared during seed maturation; their increase correlated with seed germinability after desiccation. The loss of desiccation tolerance after seed germination was also studied. For the desiccation tolerance test, intact seedlings were dried rapidly or slowly followed by rehydration. Soluble carbohydrates were assayed before and after drying. Root tissues were more sensitive to desiccation than hypocotyl tissues and completely lost desiccation tolerance within 36 h of imbibition after both fast and slow-drying treatments. Survival of hypocotyls decreased gradually up to 96 h after imbibition. Loss of RFOs and galactosyl cyclitols in axis tissues preceded visible germination. Loss of desiccation tolerance was accompanied by loss of RFOs and galactosyl cyclitols and an increase in reducing sugars in cotyledon, hypocotyl and radicle tissues. Drying did not induce the accumulation of RFOs and galactosyl cyclitols in seedling tissues.

The LEA-like protein previously isolated from a homogenate of pea (Pisum sativum L.) embryonic axes heated at 80°C for 10 min (Russouw et al., 1995) was purified without exposure to heat. Peptides produced by trypsin digestion were separated by HPLC and sequenced. The protein was identified as a member of the LEA group I family. The conformation of the protein was compared before and after heat treatment by antibody affinity, circular dichroism spectroscopy, fluorescence spectroscopy and 8-anilino-1-naphthalenesulfonic acid binding. No differences could be detected, demonstrating that the protein was not irreversibly denatured by exposure to high temperature.

Late embryogenesis abundant (LEA) proteins accumulate in developing seeds prior to maturation drying and are presumed to help protect embryos from desiccation stress. The unusual solubility properties of these proteins, such as resistance to heat coagulation, have led to suggestions that they alter the hydration properties of cellular constituents. Hydration characteristics and water potential range at which wheat heat-soluble LEA proteins were expressed have been determined. Levels of heatsoluble proteins decline in germinating seeds but can be induced by dehydration (ψ ≤ −0.5 MPa) in crown meristematic tissue that is desiccation tolerant. The heatsoluble extract from mature wheat embryos contained proteins and sugars at about a 1:1 (w/w) ratio. Only about half of the sugars could be removed by exhaustive dialysis; the rest appeared to be tightly associated with the proteins. The water sorption characteristics of undialysed and dialysed heat-soluble protein/sugar fractions were compared with other water-soluble proteins (bovine serum albumin, lysozyme or gluten) and with sucrose. At relative humidities greater than 50%, the amount of water absorbed by protein-sugar mixes was a function of the sugar content. For the same sugar content, the heat-soluble protein preparation absorbed 2–3 times more water than a lysozyme/sucrose preparation. The rate at which heat-soluble protein fractions dried was also different to desorption rates of lysozyme/sucrose mixes. While lysozyme/sucrose mixtures dried either very rapidly (within 20 min) or very slowly (about 2 months) depending on the sugar content, desorption rates of the heat soluble protein-sugar preparations were intermediate (2–10 days) and modulated by sugar concentration. Based on the presumption that the hydrophilic properties of LEA heat-soluble proteins are important to their function, it is suggested that these proteins function to control drying so that cells stay at critical water potentials for the proper time. In this respect, the heat-soluble LEA proteins do not prevent desiccation, but serve as hydration buffers.

Water contents, desiccation tolerance, respiratory rates and subcellular characteristics of three contrasting seed types were studied during development. Avicennia marina (a tropical wetland species) and Aesculus hippocastanum (a temperate species) produce recalcitrant seeds and Phaseolus vulgaris produces orthodox seeds. During development, A. hippocastanum and P. vulgaris seeds showed a decline in water content and respiration rate with a concomitant increase in desiccation tolerance. These parameters did not change during the development of A. marina seeds once they had become germinable. There was a decrease in the degree of vacuolation and an increase in the deposition of insoluble reserves in A. hippocastanum and P. vulgaris seeds, while A. marina seeds remained highly vacuolated and did not accumulate insoluble reserves. Mitochondria and endomembranes degenerated during the development of A. hippocastanum and P. vulgaris seeds, but remained unchanged in A. marina seeds. The data are consistent with the hypothesis that extensive vacuolation and high metabolic rates contribute to desiccation sensitivity. However, the development of recalcitrant A. hippocastanum seeds is similar to that of orthodox P. vulgaris seeds. These data are in accord with the concept of seed recalcitrance being a consequence of truncated development. The results suggest that there may be three categories of seeds: orthodox seeds which develop desiccation tolerance, seeds which show similar development to orthodox seeds, but are shed before desiccation tolerance is well developed, and seeds which show no developmental trends giving rise to increased tolerance.

Seeds of wild rice (Zizania palustris var. interior) have been reported to show maximum survival when dehydrated at 25°C. It has also been reported that axis cells sustain least damage at this drying temperature. In the present study, a linear relationship between drying rate and dehydration temperature was established. Whereas the highest positive tetrazolium staining and lowest leakage were recorded for seeds dehydrated at 25°C, maximum germination was recorded for seeds dried at 20°C. A proportion of seeds showed glass formation irrespective of the dehydration temperatures used. Parameters of the glass to liquid transition, however, correlated with neither water content nor sugar profiles. The ratio of raffinose to sucrose was similar in all the treatments. A hydroperoxide test revealed a linear relationship between peroxide levels and drying temperature, although fatty acid amounts were not correlated with hydroperoxide amounts. Butanal amounts and total aldehydes evolved, on the other hand, showed a high negative correlation with peroxide amounts. Electron microscopy showed that the variability and relative abundance of peripheral membrane complexes (PMCs) was highest for embryonic axes dehydrated at 25°C and lowest for cells of embryonic axes of seeds dried at 10°C. Furthermore, intramembrane particles (IMPs) were evenly distributed in cells of axes dried at 25 or 37°C. In contrast, membranes of cells of axes dehydrated at 10°C showed large IMP-free areas. The relative abundance of IMPs was highest in cells of embryonic axes dried at 25°C and lowest in cells of axes dehydrated at 10°C. From these observations, it is suggested that membrane phase transitions, with the concomitant elimination of proteins, accompany dehydration of Z. palustris seeds at 10°C, whereas at 37°C peroxidation may predominate.

Seeds of neem (Azadirachta indica A. Juss.), a multipurpose tropical tree, have limited desiccation tolerance. Whether their intermediate storage behaviour might be caused by an elevated percentage of 4C nuclei in the embryo at maturity was investigated. Morphological development of neem seeds was monitored on selected trees in Ouagadougou, Burkina Faso. Developing seeds were tested for germinability, and cell cycle events were determined by using flow cytometry and analysing the level of β-tubulin. Germination could occur after 8 weeks of seed development, but normal seedlings resulted only after 10 weeks or more. The change in fruit colour from green to yellow began after approximately 12 weeks of seed development. Immature, 4-week-old embryos about 2 mm in length had 15% of cells in the G2 phase and 60% in the G1 phase of the cell cycle, as indicated by 4C and 2C nuclear DNA levels, respectively. During maturation, the proportion of G2 cells declined to 3% at full maturity and slightly further after drying, and the proportion of G1 cells increased to approximately 90%. A strong β-tubulin signal was observed in tips of young embryonic radicles and cotyledons, but a weak or non-detectable signal was found in 9-week-old ones and in those from green-mature and yellow fruits. Because DNA replication and β-tubulin level were almost negligible at seed maturity, as in orthodox tomato seeds, it is suggested that these factors are not involved in the intermediate storage behaviour of neem seeds.

This study was undertaken to test the hypotheses that germinative metabolism of recalcitrant seeds in storage induces a requirement for additional water, which may result in the development of mild water stress, and that a reduction of the rate of this germinative metabolism will increase the storage lifespan of recalcitrant seeds. Studies were undertaken on seeds of Avicennia marina (Forssk.) Vierh. and concentrated on root primordia as these constitute the tissue that undergoes most change during storage. Encapsulating seeds from which the pericarp had been removed (naked seeds) in an alginate gel increased storage lifespan fourfold compared with naked seeds. Measures of metabolic rate such as time to first germination in storage and rate of protein synthesis did not indicate differences between alginate-coated and naked seeds, although ultrastructural observations indicated that both germinative and deteriorative processes were occurring more slowly in the alginate-coated seeds. Measures of water content and water and turgor potentials did not reveal signs of a mild water stress in either treatment. However, the number of seeds visibly contaminated with fungi and the rapidity with which this contamination became apparent were much reduced in alginate-coated seeds. It is suggested that fungal contamination constitutes a major cause of deterioration in stored, hydrated seeds of A. marina (and possibly other recalcitrant seed species) and the main effect of the alginate coating was to reduce the incidence of fungal contamination.

Mangosteen (Garcinia mangostana L.), rambai (Baccaurea motleyana Muell.- Arg.) and jelentik (Baccaurea polyneura Hook. f.) are tropical fruit species believed to have recalcitrant seeds. The seeds showed no dormancy; they germinated easily. At harvest, the mean moisture contents (fresh weight basis) were 53.54, 51.20 and 44.90% for G. mangostana, B. motleyana and B. polyneura, respectively. G. mangostana seeds lost viability when their moisture content fell to about 24% while B. motleyana seeds lost viability below 35.5% moisture content. However, for B. polyneura, the seeds could be dried to low moisture content with high survival. The viability was still high when the moisture content was reduced to 13.46%. At this moisture content, the percentage germination was 91.76% and it was found that the seeds survived cyopreservation with 8.3% viability. For B. motleyana axes, the loss of viability occurred when their moisture content fell to about 36% (15% viability with predominantly callus formation) while for B. polyneura axes, viability was reduced to 33-67% when the moisture was 27–30%. At various moisture contents, the seeds of the fruit species studied were exposed for 48 h to 7°C and −4°C. G. mangostana seeds did not survive either temperature. Baccaurea seeds survived 7°C but failed to survive −4°C. No axes from B. motleyana seeds at various moisture contents survived cryopreservation. However, some viability (20–30%) was observed in B. polyneura axes cryopreserved at a moisture content of about 27%. At this moisture content no normal growth was obtained; callus formation was observed. It appears that the seeds vary in the degree of desiccation sensitivity. They also vary in size. Seeds of G. mangostana are larger than Baccaurea seeds and thus, more sensitive to desiccation. Seed and embryonic axis structure may also play a role in desiccation sensitivity.

The flatulence-producing soluble oligosaccharides are an important component of lupin seeds and were assayed to establish the pattern of their accumulation in relation to germinability during seed development and maturation. Maturing yellow lupin cv. Juno seeds were harvested at 5-day intervals from 15 to 45 days after flowering (DAF). Seed fresh mass increased to a maximum at 35 DAF followed by a decrease when axis and cotyledon tissues changed colour from green to yellow. Maximum seed fresh mass corresponded to the maximum seed size. Seed dry mass continuously increased until 40 DAF. About 75% of mature seed dry mass was in cotyledons, 22% in testa, and 3% in axis. Maximum seed germinability occurred at 45 DAF after maximum seed dry mass and desiccation. Maturing yellow lupin seeds were desiccation tolerant. Mature dry seeds contained 10.9% oligosaccharides and 1.5% sucrose. During seed growth stachyose accumulation preceded accumulation of raffinose and verbascose. The highest rate of oligosaccharide accumulation appeared during seed desiccation and correlated with the acquisition of the ability to germinate. The sucrose:oligosaccharide ratio continuously decreased reaching 0.13 in mature seeds. Accumulation of oligosaccharides in maturing yellow lupin seeds is associated with seed germinability and seed desiccation tolerance.

Recruitment-related traits (development and dispersal of seeds, emergence, leaf dynamics, growth and survival of seedlings) were studied in Japanese elm (Ulmus davidiana var. japonica) in relation to disturbance at the soil level (bare soil, litter and control) or the canopy level (forest edge (FE), small gaps (SG) and forest understorey (FU)), or both, in a riverside forest from 1990 to 1993. Seed germination was also tested in the laboratory. Litter accumulation severely inhibited seedling emergence. Seedling emergence from a single seed rain occurred in summer immediately after seed dissemination and in the following spring. In summer, seedling emergence was higher in FE than in FU. In the following spring more seedlings emerged in FU than in FE and SG, resulting in non-significant differences in the percentage emergence among the three sites in bare soil. Darkness or far-red light induced seed dormancy, but after chilling they started to germinate again under white or red light conditions. These results explained emergence phenology under changing light conditions in the field. In FU, the spring emerging cohort grew and survived better than the summer emerging one. In FE the reverse was true. Japanese elm avoids unfavourable periods by escaping either in time (dormancy) or in space (dispersal). Such traits compensate for late and compressed seed dissemination, and improve seedling growth and survival not only when disturbance occurs at both canopy and soil level (bare soil in FE) but also only at soil level (bare soil in FU), which may occur more frequently in riverside forests.

Seeds of the narrow-endemic Solidago shortii and of the geographically-widespread S. altissima and S. nemoralis collected and buried in 1992 and 1993 were incubated in light and in darkness at 15/6°, 20/10°, 25/15°, 30/15° or 35/20°C following various periods of burial in soil in a non-temperature-controlled glasshouse. At maturity in November, seeds of the three species germinated to 0–1% in light at 15/6°C and to 10–77% at 20/10°, 25/15°, 30/15° and 35/20°C. Seeds exhumed each April from 1993 to 1996 and incubated in light at 15/6° and 20/10°C germinated to ≥83% and ≥90%, respectively, whereas those exhumed each September of 1993–96 germinated to ≤2%and ≤40%, respectively. At 25/15°, 30/15° and 35/20°C in light, seeds of S. altissima and S. shortii germinated to ≥52% and those of S. nemoralis to ≥19/, regardless of when they were exhumed. Timson's index, which integrates percentages, rates and times for onset of germination, was higher at all temperature regimes for seeds exhumed in April 1995 than for those exhumed in September 1995. Freshly-matured seeds of the three species germinated to 0–11% in darkness. Furthermore, regardless of when they were exhumed, seeds of S. altissima and S. nemoralis incubated in darkness germinated mostly to only 0–9% over the range of temperature regimes. In contrast, ≤88% and ≤6% of the seeds of S. shortii exhumed and incubated in darkness each April and September of 1993–96, respectively, germinated, ≤1% of them germinating while buried in soil. Thus, although buried seeds of all three species exhibited an annual conditional dormancy/non-dormancy cycle, only those of S. shortii exhibited cyclic changes in their germination response in darkness.