ASPECTS OF RECALCITRANT SEED PHYSIOLOGY1

N.W. PAMMENTER2 AND PATRICIA BERJAK2

School of Life and Environmental Sciences, University of Natal, Durban, South Africa ABSTRACT - Recalcitrant seeds show wide variation in water content at shedding and post-harvest physiology, particularly response to desiccation. But all are metabolically active and show germinationassociated changes in storage - it is these properties that underlie the phenomenon of recalcitrance. Three types of damage on drying are envisaged: mechanical damage associated with reduction of cellular volume; aqueous-based oxidative degradation, consequent upon unregulated metabolism, which occurs at intermediate water contents; and biophysical damage to macromolecular structures that occurs on removal of water at low water contents. The first type of damage probably occurs only in seeds with highly vacuolated cells, such as Avicennia marina, and so is probably not common. Under normal slow-drying conditions it is the second type of damage that kills recalcitrant seeds, thus the response to dehydration depends upon the metabolic activity of the seed and the rate of drying. This makes it difficult to measure desiccation tolerance - there is no such thing as a critical water content that is characteristic of a species (except the absolute minimum water content that can be tolerated on the most rapid drying possible). Probably it is the third type of damage that kills tissues that have been dried very rapidly. Desiccation sensitivity is probably the ancestral state, with tolerance having evolved independently a number of times, and recalcitrance does place constraints on the regeneration niches open to species producing such seeds. ADDITIONAL INDEX TERMS: Seed storage, desiccation tolerance, water content.

ASPECTOS DA FISIOLOGIA DE SEMENTES RECALCITRANTES

RESUMO - Sementes recalcitrantes possuem uma ampla variabilidade no contedo de gua na hora da disperso e tambm muito variabilidade na fisiologia ps-colheita, especialmente com relao resposta dessecao. Mas todas so metabolicamente ativas e mostram alteraes associadas germinao enquanto esto armazenadas so estas as propriedades que formam a base do comportamento recalcitrante. Trs tipos de injria com dessecao so visualizados: injria mecnica associada reduo do volume celular; degradao oxidativa em soluo aquosa, decorrente do metabolismo desregulada que ocorre em contedos intermedirios de gua; e dano biofsico a estruturas macromoleculares que ocorre quando se remove gua em contedos muito baixos. O primeiro tipo de injria provavelmente ocorre somente em sementes com clulas altamente vacuoladas, como as de Avicennia marina, e no deve ser muito comum. Em condies normais de secagem lenta, o segundo tipo de injria que leva morte das sementes recalcitrantes, assim a resposta desidratao depende da atividade metablica da semente e da taxa de dessecamento. Desta maneira difcil medir a tolerncia dessecao no existe um contedo crtico de umidade que caracteriza a espcie (a no ser o contedo mnimo absoluto que possa ser tolerado com
1. Based on an invited lecture presented at the VII Brazilian Plant Physiology Congress, Braslia, July, 1999, in the Coordinated Session on Seed Physiology 2. School of Life and Environmental Sciences, George Campbell Building, University of Natal, Durban, 4014 South Africa. E-mail: pammenter@biology.und.ac.za.

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57

a secagem o mais rpido possvel). Provavelmente o terceiro tipo de injria que letal para tecidos que foram dessecados muito rapidamente. A sensitividade dessecao provavelmente o estado ancestral, e a tolerncia evoluiu vrias vezes, de maneira independente. Existem limitaes quanto aos nichos ecolgicos nos quais as espcies que produzem sementes recalcitrantes podem se regenerar. TERMOS ADICIONAIS PARA INDEXAO: Armazenamento de sementes, contedo de umidade, tolerncia dessecao.

INTRODUCTION Orthodox seeds are seeds that acquire desiccation tolerance during development, can dry to low water contents (generally less than 5%), and retain viability in the dry state for predictable periods. Recalcitrant seeds, on the other hand, are shed at high water contents, ranging from 0.4 to 4.0 g water per g dry matter (g/g), are sensitive to desiccation, and are also metabolically active on shedding. A characteristic of recalcitrant seeds is the variability they show among species and within a species. They vary in the water content at shedding, the extent of dehydration they tolerate, their response to drying rate, storage lifespan in the hydrated state (from a week or two, to two or three years), and their response to low temperatures (for some examples see King and Roberts, 1980; Farrant et al., 1985, 1986, 1989; Hong and Ellis, 1990; Pritchard, 1991; Berjak et al., 1992, 1993; Finch-Savage, 1992; Tompsett, 1992; Xia et al., 1992; Tompsett and Pritchard, 1993, 1998). This means that it is not simply a case of classifying a seed as orthodox or recalcitrant, but within the recalcitrant group there is a wide spectrum of behaviours, from minimally recalcitrant seeds with a relatively long lifespan and quite tolerant of desiccation, to maximally recalcitrant, with short lifespans and very sensitive to desiccation (Farrant et al., 1988).

PROPERTIES OF WATER IN SEEDS Before considering the response of recalcitrant seeds to the loss of water, we should consider the properties of water in seed tissue (see Vertucci, 1990; Vertucci and Farrant, 1995; Walters, this volume). Based on calorimetric properties, Vertucci (1990) has identified five types of water or levels of hydration in seed tissues. The different hydration levels correspond roughly to the water contents shown in Table 1, and the water potentials at the boundaries are also indicated. At the different hydration levels the tissue water has different physical properties. At high water contents and high water potential the water has the properties of water in dilute solution. As the water content is decreased the water takes on the properties of water in concentrated solution, where the interaction between water and solutes becomes stronger, and the system deviates from "ideal" behaviour. On the removal of more water, the solution becomes so concentrated that it becomes viscous and has the properties of a glass. Finally, at very low water contents, those characteristic of orthodox seeds, all the remaining water is tightly associated with macromolecular surfaces; its mobility is reduced and it constitutes the so-called 'bound' water. At the different hydration levels, because the thermodynamic properties of water change, different metabolic processes can take place

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TABLE 1 - The properties of water in seed tissue and different hydration levels. The approximate water content range, water potential corresponding to the boundaries, and the type of metabolic activity that occurs are also given. Adapted from Vertucci and Farrant (1995).
Hydration Level Water Content (g/g) <0.08 0.08 - 0.25 0.25 - 0.45 0.45 - 0.70 > 0.70 I II III IV V

Water potential (MPa)

-150

-11/-14

-3

-1.5

State of water

bound

glass/ hydrophilic

glass/ hydrophobic

Concentrated solution

Dilute Solution

Activity

low level catabolic

respiration

protein and nucleic acid synthesis

cell division: germination

(Vertucci and Leopold, 1986; Vertucci, 1989; Vertucci and Farrant, 1995). At the high water contents, full normal metabolism occurs and seeds can germinate. In hydration level IV, protein and nucleic acid synthesis, together with respiration, is possible, but there is inadequate water for cell growth and germination. At lower water contents protein and nucleic acid synthesis are not possible, but some respiration can be detected. At even lower water contents only low level catabolic events occur slowly. DEVELOPMENT IN ORTHODOX AND RECALCITRANT SEEDS Before considering the response of recalcitrant seeds to water loss, it would be useful to consider the processes occurring when orthodox seeds undergo maturation drying. As the seeds dry, insoluble reserves accumulate, the volume of water-filled vacuoles is reduced, and protective

molecules are synthesized. These modifications are followed, or accompanied, by the de-differentiation of highly structured organelles, particularly mitochondria, and a general 'switching-off' of metabolism, until finally the protective mechanisms such as vitrification and the deployment of Late Embryogenic Abundant (LEA) proteins become operative. Throughout the drying process, presumably, anti-oxidant mechanisms are operative. There are limited data from orthodox seeds, but certainly anti-oxidant activity is important during drying of desiccation-tolerant resurrection" plants (reviewed by Oliver and Bewley, 1997). To summarise, during the later stages of development of orthodox seeds the processes of reserve accumulation, reduction of vacuolar volume, de-differentiation and shut-down of metabolism accompany the acquisition of desiccation tolerance. The question then is whether these processes are necessary for the acquisition of

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59 viability loss. Thus there is a sequence of sensitivities: Avicennia > Aesculus > Phaseolus. Figure 1a shows the contribution of vacuoles to cell volume for the three stages averaged over the cotyledon, hypocotyl and root meristem. We see quite clearly that Avicennia marina, the most sensitive species, was highly vacuolated, and this did not change with development, while the more tolerant species showed a decline in vacuolation with development. The inverse pattern is seen with insoluble reserves (Figure 1b): Avicennia marina, the most sensitive species, stores its reserves as soluble sugars, while the more tolerant species accumulated insoluble reserves during development. The metabolic activity of the seeds is also pertinent. In the orthodox Phaseolus vulgaris there was a decline in the contribution of mitochondria to cell volume with development, and by the end of reserve accumulation, mitochondria were virtually absent (Figure 1c). Although there was a decline in seeds of Aesculus hippocastanum, by the time the seeds were shed mitochondria still contributed significantly to cell volume. However, it is not only the quantitative contribution of mitochondria that is important to metabolic activity, it is also the qualitative degree of differentiation.

tolerance, and whether and to what extent they occur in recalcitrant seeds. To answer this question we studied the development of three very different seed types: Avicennia marina, which is a mangrove, a tropical wetland species which produces highly recalcitrant seeds; Aesculus hippocastanum, a temperate terrestrial species producing recalcitrant seeds; and Phaseolus vulgaris, an agricultural species that produces orthodox seeds (Farrant et al., 1997). We studied these seeds at three developmental stages: immediately post histodifferentiation (stage 1), in the middle of the reserve accumulation phase (stage 2), and at the end of reserve accumulation (stage 3; P. vulgaris seeds were still at a high water content at this stage; it was before maturation drying). Table 2 shows the desiccation sensitivity of seeds of the three species at the three developmental stages (measured as the water content at which the viability of rapidly dried excised axes was lost). Avicennia marina is very sensitive and tolerance did not change from stage 2 to stage 3. Aesculus hippocastanum showed an increase in tolerance with development, but at shedding was still sensitive. Phaseolus vulgaris increased in tolerance with development, and during maturation drying became even more tolerant, achieving water contents of less than 0.08 g/g without

TABLE 2 - Desiccation sensitivity of Avicennia marina, Aesculus hippocastanum and Phaseolus vulgaris at three stages of development, as measured by the water content (g/g) of isolated embryonic axes at which viability was zero after drying as rapidly as possibly. [Stage 1: post-histodifferentiation; stage 2: in the middle of reserve deposition; stage 3: at the end of reserve deposition (prior to maturation drying in the case of P. vulgaris)]. Stage 1 2 3 A. marina 0.5 0.5 0.5 A. hippocastanum 3.5 0.8 0.3 P. vulgaris 1.5 1.5 0.3

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60

a
50

stage 1

stage 2

stage 3

Area Vacuoles (%)

40 30 20 10 0

60

Area Reserves (%)

50 40 30 20 10 0

2,0 Area Mitochondria (%) 1,5 1,0 0,5 0,0

A. m ar in a

A. hi pp oc as ta nu m

FIGURE 1 - The contribution of vacuoles (a), insoluble reserves (b) and mitochondria (c) to cells of seeds of Avicennia marina, Aesculus hippocastum and Phaseolus vulgaris at three developmental stages (see text for details). Contribution was estimated as the percentage of cell cross-sectional area occupied by each type of organelle, averaged over the cotyledons, hypocotyl and root meristems.
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P. vu lg ar is

Aspects of recalcitrant ...

61 In summary, orthodox seeds show a fixed developmental pattern in that, after reserve accumulation, they acquire desiccation tolerance and undergo maturation drying. During the development of most recalcitrant seeds there is some decline in water content (but mostly because dry matter accumulates faster than water, rather than water loss) and some increase in desiccation tolerance. It has been suggested that recalcitrant seeds show an indeterminate developmental pattern in that development is truncated and the seeds are shed before desiccation tolerance is fully acquired (e.g. Finch-Savage and Blake, 1994). Certainly, the lethal water content that one measures depends upon the developmental status of the seed. Post-shedding development also has an effect on desiccation sensitivity. The longer seeds are stored, the more sensitive they become (Farrant et al., 1986; Berjak et al., 1992, 1993). We attribute this observation to the fact that germination is initiated during storage (thus metabolism is increasing), and suggest that the effect of developmental stage on desiccation sensitivity is related to metabolic rate. Throughout pre-and post-shedding development, desiccation sensitivity changes in parallel with metabolic rate.

Transmission electron microscopy showed that the mitochondria of the root meristem cells of Aesculus hippocastanum were highly differentiated and had the appearance of active mitochondria, while those in Phaseolus were de-differentiated and appeared inactive (see Berjak and Pammenter, this volume). Measured respiration rates support the ultrastructural observations, remaining high in the recalcitrant seeds Avicennia marina and Aesculus hippocastanum throughout development, while the respiration rates of the orthodox seeds of Phaseolus vulgaris were low, even though the seeds had not gone through maturation drying and were still hydrated (Table 3). Thus, the most desiccation sensitive species is the one that remained highly vacuolated, supporting the concept that drying of highly vacuolated material leads to mechanical damage. Also, reduction in metabolic rate and dedifferentiation appear to be prerequisites for survival of dehydration. A characteristic of all recalcitrant seeds we have studied is that they are metabolically active; they show high rates of respiration relative to orthodox seeds, and have ultrastructure that is characteristic of metabolically active tissue.

TABLE 3 - Respiration rates (nmol O2 (g dry mass)-1 s-1) of cotyledons and embryonic axes of Avicennia marina, Aesculus hippocastanum and Phaseolus vugaris at three stages of development. [Stage 1: posthistodifferentiation; stage 2: in the middle of reserve deposition; stage 3: at the end of reserve deposition (prior to maturation drying in the case of P. vulgaris)]. A. marina Stage 1 2 3 Cotyledons 5.4 4.5 4.6 Axes A. hippocastanum Cotyledons 5.0 3.9 3.0 Axes 5.3 5.0 4.7 P. vulgaris Cotyledons 3.5 0.9 0.2 Axes 1.8 1.1

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62 OTHER PROTECTION MECHANISMS During the final stages of development of orthodox seeds there is the reduction in the volume of water-filled vacuoles, the de-differentiation of organelles and the shut down of metabolism that are necessary for desiccation tolerance. However, other processes, involving particularly Late Embryogenic Abundant (LEA) proteins and sugars, also occur. LEA proteins accumulate during the later stages of embryogenesis in orthodox seeds (Galau et al., 1986, 1991). These proteins are similar to the dehydrins produced on drying of vegetative tissue. In fact, dehydrins are a subset of LEAs. These proteins are thought to have a protective role in the dry state, or certainly to contribute in some way to desiccation tolerance (reviewed by Oliver and Bewley, 1997; Kermode, 1997), although details of their mode of action are not yet clear. LEAs have been shown to be present in quite a wide range of recalcitrant seeds, mostly temperate (Bradford and Chandler, 1992; FinchSavage et al., 1994a), but also some tropical species (Gee et al., 1994; Farrant et al., 1996). However, they are not universally present in all recalcitrant seeds; they are certainly absent from the axes of several tropical wetland species (Farrant et al., 1996). It is possible that the presence of LEAs can contribute to the slightly greater desiccation tolerance and chilling tolerance of some recalcitrant species. If sucrose and oligosaccharides such as raffinose are present in the correct ratios, then a glass can be formed as water is lost. This state is highly viscous, which should slow down any chemical processes occurring in the seed, thus providing protection on desiccation (Williams and Leopold, 1989; Leopold et al., 1994). Sugars, possibly in the appropriate mass ratios, occur in many recalcitrant seeds (Farrant et al., 1993;

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Finch-Savage and Blake, 1994; Lin and Huang, 1994; Steadman et al., 1996), and it is possible that glasses may form on dehydration. However, these glasses, if they do form, are obviously not effective protectants, as dehydration does kill recalcitrant seeds. To summarise, some species of recalcitrant seeds do have the appropriate sugars and some have dehydrins. However, it should be pointed out that these mechanisms are thought to operate and to provide protection at low water contents. On dehydration recalcitrant seeds die at water contents higher than those at which these protective measures are thought to operate. Whatever the role of LEAs and sugars may be in orthodox seeds, they are not pertinent to recalcitrant seeds. It is now relatively well established that treatments which disturb normal metabolism can generate free radicals, and protection against these free radicals by anti-oxidant systems is essential for survival. Highly active anti-oxidant systems are certainly very important in the protection mechanisms of resurrection plants, both on drying and during rehydration (Oliver and Bewley, 1997). Although not a lot of work has been done, these mechanisms are present in orthodox seeds. During the drying of recalcitrant seeds, stable free radicals do accumulate (Hendry et al., 1992; Finch-Savage et al., 1993, 1994b). There is evidence that anti-oxidant systems also fail (Hendry et al., 1992; Finch-Savage et al., 1993), or they may not be present at sufficient levels. Oxidative damage probably does occur during drying. There have been some new suggestions concerning the mechanisms of desiccation tolerance (Hoekstra et al., 1997; Golovina et al., 1998). These authors postulate the existence of amphipathic molecules that become integrated into the membrane as water is lost. It is suggested that these molecules lower the water content at which

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63 thus for rapid drying the embryonic axis has been removed from the influence of the rest of the seed. To resolve this question we undertook studies on whole seeds of Ekebergia capensis (Pammenter et al., 1998). This species produces seeds about the size of a peanut with a hard endocarp. If the endocarp is removed the seeds can be dried by burying in silica gel to an axis water content of around 0.2 g/g (dry mass basis) in about 48 h (Figure 2a). If the endocarp is left intact it takes about 10 d for axis water content to reduce to 0.5 g/g (Figure 2b). We also manipulated the drying technique to achieve what we describe as an intermediate drying rate. The response of final germination to water content using the three drying treatments is seen in Figure 3. Seeds that were dried slowly started to lose viability at an axis water content of about 1.3 g/g and were all dead at 0.6 g/g. Seeds that were dried rapidly retained 90% viability to a water content of 0.5 g/g, and were all dead at 0.2 g/g. Seeds dried at an intermediate rate showed an intermediate response to desiccation. The rate of drying does have a marked effect on the water content tolerated; the effect is not one of removing the axis from the seed. Why should this be the case? The cells of embryonic axes of the seeds dried at different rates showed very different ultrastructural responses to dehydration (see Berjak and Pammenter, this volume). In seeds that were dried slowly to the extent that considerable viability loss occurred, there was advanced degradation of membrane structures, particularly in the plastids, and an abnormal appearance of the lipid bodies, particularly a darkening of the surface. Seeds that had been dried rapidly to a slightly lower water content, but still were mostly viable, generally showed well preserved membranes and nuclei. Although membrane damage did ultimately occur on rapid drying, there

lipid phase changes occur and thus possibly stabilize the integral proteins in the membranes. This is recent work and so far no information is available with regard to the presence of these molecules in recalcitrant seeds. It is apparent that there is a suite, a collection, of mechanisms that are thought to protect against desiccation damage. Some of these protective mechanisms probably also operate during rehydration, as well as during drying. The different protective mechanisms seem to operate in different water content ranges. All these mechanisms must be operational for desiccation tolerance. Recalcitrant seeds lack some or all of these mechanisms. It is not apparent whether there is any relationship between the degree of recalcitrance of a seed and the extent to which it has or expresses some of these mechanisms. THE IMPORTANCE OF DEHYDRATION RATE Dehydration rate is perhaps one of the most confusing issues in recalcitrant seed physiology. When whole seeds are dried relatively slowly - over several days - viability is generally lost at embryonic axis water contents in the range of 1.0 - 0.5 g water/g dry mass (50 - 35% wet mass basis). However, if isolated embryonic axes are dried rapidly (minutes to hours) they survive to much lower water contents - 0.45 to 0.25 g/g - and possibly even lower (e.g. Pammenter et al., 1991). Thus the effect of dehydration depends upon the rate of dehydration. This makes it extremely difficult to assess or measure desiccation tolerance - the response depends upon how the tissue is dried. However, this approach could be criticised because of the experimental protocol; the effect could be due to different drying rates, or it could be due to the fact that different tissues were used to achieve the different drying rates (embryonic axes for rapid drying and whole seeds for slow drying);

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64 was very marked degradation at high water contents in slowly dried material. This ultrastructural evidence suggests that different deteriorative processes may be occurring at the different drying rates. To understand the effect of drying rate we have to appreciate that some damaging processes are aqueous based. Slowly dried material will spend a long time at the intermediate water contents where damage from
2,0

Pammenter and Berjak

these aqueous based processes can accumulate. However, if material is dried rapidly, only a short time is spent at these intermediate water contents, so little damage accumulates. Thus the faster the drying, the less damage that accumulates and the lower the water content that can be tolerated. However, there is a lower limit - no matter how fast recalcitrant seeds are dried, they cannot tolerate the low water contents typical of orthodox seeds.

a
Axis water content (g/g) 1,5

1,0

0,5

0,0 0 20 40 Drying time (h) 60 80

2,5 Axis water content (g/g) 2,0 1,5 1,0 0,5 0,0 0 2 4 6 8 10 Drying time (d) slow intermediate

FIGURE 2 -The drying time course of embryonic axes of Ekebergia capensis when whole seeds were dried either rapidly (4a) or at an intermediate or slow rate (4b).

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65

100 80 60 40 20 0 0,0 0,5 1,0 1,5 2,0 2,5 Axis water content (g/g)

Germination (%)

rapid slow intermediate

FIGURE 3 - Final germination of seeds of Ekebergia capensis dried at different rates to a range of axis water contents. We visualise two types of damage that seed desiccation response as a character state fails can occur during drying. As a consequence of both tolerance and sensitivity occur in all major disturbed metabolism, free radical-mediated clades of extant seed plants. However, it is oxidation processes can proceed. These reactions are probable that sensitivity is the ancestral state, but aqueous-based and occur at intermediate water tolerance evolved independently a number of times contents. The protection mechanisms against this (von Teichman and van Wyk, 1994; Pammenter and type of damage are the switching-off of metabolism, de-differentiation of organelles, and efficient anti- Berjak, 2000). This raises the interesting question as oxidant systems. The second type of damage we to whether the mechanisms of tolerance are envisage is due to the biophysical disruptions caused necessarily the same in all seed species. by the removal of water from macromolecular and ECOLOGICAL CONSIDERATIONS membrane surfaces. These occur at low water contents. Protection mechanisms against this type of A characteristic of recalcitrant seeds is damage include LEAs, vitrification by sugars, and possibly the amphipathic molecules their limited lifespan, and this will place suggested to migrate between cytosol and constraints on the range of environmental membranes on dehydration and rehydration. conditions in which regeneration via seeds can occur. However, when we try to get detailed EVOLUTIONARY STATUS OF information on the seed ecology of species producing recalcitrant seeds, we have a major RECALCITRANT SEEDS problem in the past ecologists and seed scientists In vegetative tissue desiccation have not often communicated. Ecologists study the sensitivity is probably the ancestral state, but composition of soil seed banks, the lifespan of tolerance evolved independently a number of seeds in these banks, and environmental conditions times, including more than once in the initiating germination, but often have no angiosperms (Oliver and Bewley, 1997). Any information as to the water content of the seeds attempt at a similar phylogenetic analysis using of interest. Seed physiologists, on the other
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66 hand, have a good knowledge of only a few species, mostly economically important ones (either crop species or weedy invaders), and only occasionally become involved in ecological considerations. Recalcitrant seeds are common in mesic tropical forests. These conditions generally would be favourable for germination and seedling establishment, and so there has been no pressure driving the evolution of desiccation tolerance, or the characteristic has been secondarily lost. Seed ecologists will tell us that seeds of climax species of these ecosystems are not often found in the soil seed bank their lifespans are low, whereas seeds of gap specialists are well represented in the soil seed bank. The seeds of climax species generally show no dormancy, germinate rapidly (unless they have a hard covering), are absent from the soil seed bank and these species persist as a seedling bank. They are generally large and not wind dispersed. These are the characteristics that one would expect from recalcitrant seeds, and it is highly probable that most of these seed species are indeed recalcitrant, although this has been confirmed for only a limited number of species. Although recalcitrant seeds are common in mesic tropical forests, they are by no means confined to these systems. Species producing recalcitrant seeds do occur in more seasonal habitats, and a number of strategies have evolved to permit successful seedling establishment. Many temperate recalcitrant seeds are shed in autumn and over-winter as seeds. A necessary prerequisite for this, of course, is chilling tolerance. Because of the low temperatures, rates of germination will be slow, and will not be completed before the arrival of spring. In fact some species such as Aesculus hippocastanum actually have a chilling requirement for germination (Pritchard et al., 1996). Low temperatures and general dampness will also slow water loss, reducing the risk of death by desiccation. As a generalisation, recalcitrant

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species of temperate regions are generally more desiccation tolerant and have longer life spans than those of tropical origin.

REFERENCES BERJAK, P.; PAMMENTER, N.W. & VERTUCCI, C.W. Homoiohydrous (recalcitrant) seeds: developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta, 186:249-261,1992. BERJAK, P.; VERTUCCI, C.W. & PAMMENTER, N W. Effects of developmental status and dehydration rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia sinensis. Seed Science Research, 3:155-166, 1993. BRADFORD, K.J. & CHANDLER, P.M. Expression of dehydrin-like proteins in embryos and seedlings of Zizania palustris and Oryza sativa during dehydration. Plant Physiology, 99:488-494, 1992. FARRANT, J.M.; BERJAK, P. & PAMMENTER, N.W. The effect of drying rate on viability retention of recalcitrant propagules of Avicennia marina. South African Journal of Botany, 51:432-438, 1985. FARRANT, J.M.; PAMMENTER, N.W. & BERJAK, P. The increasing desiccation sensitivity of recalcitrant Avicennia marina seeds with storage time. Physiologia Plantarum, 67:291-298, 1986. FARRANT, J.M.; PAMMENTER, N.W. & BERJAK, P. Recalcitrance - a current assessment. Seed Science and Technology, 16:155-166, 1988.

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67 FINCH-SAVAGE, W.E.; HENDRY, G.A.F. & ATHERTON, N.M. Free radical activity and loss of viability during drying of desiccationsensitive tree seeds. Proceedings of the Royal Society of Edinburgh, 102B:257260, 1994b. FINCH-SAVAGE, W.E.; PRAMANIK, S.K. & BEWLEY, J.D. The expression of dehydrin proteins in desiccation-sensitive (recalcitrant) seeds of temperate trees. Planta, 193:478-485, 1994a. GALAU, G.A.; HUGHES, D.W. & DURE, L. Abscisic acid induction of cloned cotton late embryogenesis-abundant (lea) mRNAs. Plant Molecular Biology, 7:157-170, 1986. GALAU, G.A.; JAKOBSEN, K.S. & HUGHES, D.W. The controls of late dicot embryogenesis and early germination. Physiologia Plantarum, 81:280-288, 1991. GEE, O.H.; PROBERT, R.J. & COOMBER, S.A. 'Dehydrin-like' proteins and desiccation tolerance in seeds. Seed Science Research, 4:135-141, 1994. GOLOVINA, E.A.; HOEKSTRA, F.A. & HEMMINGA, M.A. Drying increases intracellular partioning of amphiphilic substances into the lipid phase: impact on membrane permeability and significance for desiccation tolerance. Plant Physiology, 118:975-986, 1998. HENDRY, G.A.F.; FINCH-SAVAGE, W.E.; THORPE, P.C.; ATHERTON, N.M.; BUCKLAND, S.M.; NILSSON, K.A. & SEEL, W.A. Free radical processes and loss of viability during desiccation in the recalcitrant species Quercus robur L. New Phytologist, 122:273-279, 1992.

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