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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-harvestphysiology, particularly response to desiccation. But all are metabolically active and show germination-associated changes in storage - it is these properties that underlie the phenomenon of recalcitrance. Threetypes of damage on drying are envisaged: mechanical damage associated with reduction of cellularvolume; aqueous-based oxidative degradation, consequent upon unregulated metabolism, which occurs atintermediate water contents; and biophysical damage to macromolecular structures that occurs on removalof water at low water contents. The first type of damage probably occurs only in seeds with highlyvacuolated cells, such asAvicennia marina, and so is probably not common. Under normal slow-dryingconditions it is the second type of damage that kills recalcitrant seeds, thus the response to dehydrationdepends upon the metabolic activity of the seed and the rate of drying. This makes it difficult to measuredesiccation 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. Desiccationsensitivity 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 dadisperso e tambm muito variabilidade na fisiologia ps-colheita, especialmente com relao resposta dessecao. Mas todas so metabolicamente ativas e mostram alteraes associadas germinaoenquanto 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 volumecelular; degradao oxidativa em soluo aquosa, decorrente do metabolismo desregulada que ocorre emcontedos intermedirios de gua; e dano biofsico a estruturas macromoleculares que ocorre quando seremove gua em contedos muito baixos. O primeiro tipo de injria provavelmente ocorre somente emsementes com clulas altamente vacuoladas, como as deAvicennia marina, e no deve ser muito comum.Em condies normais de secagem lenta, o segundo tipo de injria que leva morte das sementesrecalcitrantes, assim a resposta desidratao depende da atividade metablica da semente e da taxa dedessecamento. Desta maneira difcil medir a tolerncia dessecao no existe um contedo crticode 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 CoordinatedSession on Seed Physiology

2. School of Life and Environmental Sciences, George Campbell Building, University of Natal, Durban, 4014 South Africa.E-mail: [email protected].


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Aspects of recalcitrant ... 57

R. Bras. Fisiol. Veg., 12(Edio Especial):56-69, 2000

a secagem o mais rpido possvel). Provavelmente o terceiro tipo de injria que letal para tecidos queforam dessecados muito rapidamente. A sensitividade dessecao provavelmente o estado ancestral, ea 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 acquiredesiccation tolerance during development, can dryto low water contents (generally less than 5%), and

retain viability in the dry state for predictableperiods. Recalcitrant seeds, on the other hand, areshed at high water contents, ranging from 0.4 to4.0 g water per g dry matter (g/g), are sensitive todesiccation, and are also metabolically active onshedding.

A characteristic of recalcitrant seeds isthe variability they show among species and withina species. They vary in the water content atshedding, the extent of dehydration they tolerate,

their response to drying rate, storage lifespan in thehydrated state (from a week or two, to two or threeyears), 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). Thismeans 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 ofbehaviours, from minimally recalcitrant seeds witha relatively long lifespan and quite tolerant ofdesiccation, to maximally recalcitrant, with shortlifespans and very sensitive to desiccation (Farrant

et al., 1988).

PROPERTIES OF WATER IN SEEDS

Before considering the response ofrecalcitrant seeds to the loss of water, we shouldconsider the properties of water in seed tissue (see

Vertucci, 1990; Vertucci and Farrant, 1995;Walters, this volume). Based on calorimetricproperties, Vertucci (1990) has identified five typesof water or levels of hydration in seed tissues. Thedifferent hydration levels correspond roughly tothe water contents shown in Table 1, and the waterpotentials at the boundaries are also indicated. Atthe different hydration levels the tissue water hasdifferent physical properties. At high watercontents and high water potential the water has the

properties of water in dilute solution. As the watercontent is decreased the water takes on theproperties of water in concentrated solution, where

the interaction between water and solutes becomesstronger, and the system deviates from "ideal"

behaviour. On the removal of more water, thesolution becomes so concentrated that it becomes

viscous and has the properties of a glass. Finally, atvery low water contents, those characteristic of

orthodox seeds, all the remaining water is tightly

associated with macromolecular surfaces; itsmobility is reduced and it constitutes the so-called'bound' water.

At the different hydration levels, becausethe thermodynamic properties of water change,different metabolic processes can take place


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58 Pammenter and Berjak


TABLE 1 - The properties of water in seed tissue and different hydration levels. The approximate watercontent range, water potential corresponding to the boundaries, and the type of metabolic activity thatoccurs are also given. Adapted from Vertucci and Farrant (1995).

Hydration

Level

I II III IV V

WaterContent

(g/g)

0.70

Waterpotential

(MPa)

-150 -11/-14 -3 -1.5

State ofwater bound glass/hydrophilic glass/hydrophobic Concentratedsolution DiluteSolution

Activity low levelcatabolic

respiration protein andnucleic acid

synthesis

cell division:germination

(Vertucci and Leopold, 1986; Vertucci, 1989;Vertucci and Farrant, 1995). At the high watercontents, full normal metabolism occurs and seedscan germinate. In hydration level IV, protein andnucleic acid synthesis, together with respiration, ispossible, but there is inadequate water for cellgrowth and germination. At lower water contentsprotein and nucleic acid synthesis are not possible,but some respiration can be detected. At evenlower water contents only low level catabolicevents occur slowly.

DEVELOPMENT IN ORTHODOX ANDRECALCITRANT SEEDS

Before considering the response ofrecalcitrant seeds to water loss, it would be usefulto consider the processes occurring when orthodoxseeds undergo maturation drying. As the seeds dry,insoluble reserves accumulate, the volume ofwater-filled vacuoles is reduced, and protective

molecules are synthesized. These modifications arefollowed, or accompanied, by the de-differentiationof highly structured organelles, particularlymitochondria, and a general 'switching-off' ofmetabolism, until finally the protectivemechanisms such as vitrification and thedeployment of Late Embryogenic Abundant (LEA)proteins become operative. Throughout the dryingprocess, presumably, anti-oxidant mechanisms areoperative. There are limited data from orthodoxseeds, but certainly anti-oxidant activity isimportant during drying of desiccation-tolerantresurrection" plants (reviewed by Oliver andBewley, 1997).

To summarise, during the later stages ofdevelopment of orthodox seeds the processes ofreserve accumulation, reduction of vacuolarvolume, de-differentiation and shut-down ofmetabolism accompany the acquisition ofdesiccation tolerance. The question then is whetherthese processes are necessary for the acquisition of


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tolerance, and whether and to what extent theyoccur in recalcitrant seeds.

To answer this question we studied the

development of three very different seed types:Avicennia marina, which is a mangrove, a tropicalwetland species which produces highlyrecalcitrant seeds; Aesculus hippocastanum, atemperate terrestrial species producingrecalcitrant seeds; and Phaseolus vulgaris, anagricultural species that produces orthodox seeds(Farrant et al., 1997). We studied these seeds atthree developmental stages: immediately posthistodifferentiation (stage 1), in the middle ofthe reserve accumulation phase (stage 2), and atthe end of reserve accumulation (stage 3; P.vulgaris seeds were still at a high water content atthis stage; it was before maturation drying).

Table 2 shows the desiccation sensitivityof seeds of the three species at the threedevelopmental stages (measured as the watercontent at which the viability of rapidly driedexcised axes was lost). Avicennia marina is verysensitive and tolerance did not change from stage 2to stage 3. Aesculus hippocastanum showed anincrease in tolerance with development, but atshedding was still sensitive. Phaseolus vulgarisincreased in tolerance with development, and duringmaturation drying became even more tolerant,achieving water contents of less than 0.08 g/g without

viability loss. Thus there is a sequence ofsensitivities: Avicennia > Aesculus > Phaseolus.

Figure 1a shows the contribution ofvacuoles to cell volume for the three stagesaveraged over the cotyledon, hypocotyl and rootmeristem. We see quite clearly that Avicenniamarina, the most sensitive species, was highlyvacuolated, and this did not change withdevelopment, while the more tolerant speciesshowed a decline in vacuolation with development.The inverse pattern is seen with insoluble reserves(Figure 1b): Avicennia marina, the most sensitivespecies, stores its reserves as soluble sugars, while

the more tolerant species accumulated insolublereserves during development.

The metabolic activity of the seeds is alsopertinent. In the orthodoxPhaseolus vulgaris therewas a decline in the contribution of mitochondriato cell volume with development, and by the end ofreserve accumulation, mitochondria were virtuallyabsent (Figure 1c). Although there was a decline inseeds of Aesculus hippocastanum, by the timethe seeds were shed mitochondria stillcontributed significantly to cell volume.However, it is not only the quantitativecontribution of mitochondria that is importantto metabolic activity, it is also the qualitativedegree of differentiation.

TABLE 2 -Desiccation sensitivity ofAvicennia marina, Aesculus hippocastanumandPhaseolus vulgarisat three stages of development, as measured by the water content (g/g) of isolated embryonic axes atwhich viability was zero after drying as rapidly as possibly. [Stage 1: post-histodifferentiation; stage 2: inthe middle of reserve deposition; stage 3: at the end of reserve deposition (prior to maturation drying inthe case ofP. vulgaris)].

Stage A. marina A. hippocastanum P. vulgaris

1 0.5 3.5 1.5

2 0.5 0.8 1.5

3 0.5 0.3 0.3


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FIGURE 1 - The contribution of vacuoles (a), insoluble reserves (b) and mitochondria (c) to cells ofseeds ofAvicennia marina, Aesculus hippocastumandPhaseolus vulgaris at three developmental stages(see text for details). Contribution was estimated as the percentage of cell cross-sectional area occupiedby each type of organelle, averaged over the cotyledons, hypocotyl and root meristems.

0

10

20

30

40

50

60

AreaVacuoles(%)

s tage 1 stage 2 stage 3a

0

10

20

30

40

50

60

AreaReserves(%)

0,0

0,5

1,0

1,5

2,0

A.marina

A.hippocas

tanu

m

P.vulg

aris

AreaMitochondria(%)

c

b


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Transmission electron microscopyshowed that the mitochondria of the rootmeristem cells of Aesculus hippocastanum were

highly differentiated and had the appearanceof active mitochondria, while those in Phaseoluswere de-differentiated and appeared inactive(see Berjak and Pammenter, this volume).Measured respiration rates support theultrastructural observations, remaining high inthe recalcitrant seeds Avicennia marina and

Aesculus hippocastanum throughoutdevelopment, while the respiration rates of theorthodox seeds of Phaseolus vulgaris were low,even though the seeds had not gone throughmaturation drying and were still hydrated(Table 3).

Thus, the most desiccation sensitivespecies is the one that remained highly vacuolated,supporting the concept that drying of highlyvacuolated material leads to mechanical damage.Also, reduction in metabolic rate and de-differentiation appear to be prerequisites forsurvival of dehydration. A characteristic of allrecalcitrant seeds we have studied is that they aremetabolically active; they show high rates ofrespiration relative to orthodox seeds, and haveultrastructure that is characteristic of metabolicallyactive tissue.

In summary, orthodox seeds show afixed developmental pattern in that, after reserveaccumulation, they acquire desiccation tolerance

and undergo maturation drying. During thedevelopment of most recalcitrant seeds there issome decline in water content (but mostlybecause dry matter accumulates faster thanwater, rather than water loss) and someincrease in desiccation tolerance. It has beensuggested that recalcitrant seeds show anindeterminate developmental pattern in thatdevelopment is truncated and the seeds are shedbefore desiccation tolerance is fully acquired (e.g.Finch-Savage and Blake, 1994). Certainly, thelethal water content that one measures dependsupon the developmental status of the seed.

Post-shedding development also has aneffect on desiccation sensitivity. The longerseeds are stored, the more sensitive they become(Farrant et al., 1986; Berjak et al., 1992, 1993).We attribute this observation to the fact thatgermination is initiated during storage (thusmetabolism is increasing), and suggest that theeffect of developmental stage on desiccationsensitivity is related to metabolic rate.Throughout pre-and post-shedding development,desiccation sensitivity changes in parallel withmetabolic rate.

TABLE 3 -Respiration rates (nmol O2(g dry mass)-1s-1) of cotyledons and embryonic axes of Avicennia

marina, Aesculus hippocastanumand Phaseolus vugaris at three stages of development. [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 ofP. vulgaris)].

A. marina A. hippocastanum P. vulgaris

Stage Cotyledons Axes Cotyledons Axes Cotyledons Axes

1 5.4 - 5.0 5.3 3.5 -

2 4.5 - 3.9 5.0 0.9 1.8

3 4.6 - 3.0 4.7 0.2 1.1


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OTHER PROTECTION MECHANISMS

During the final stages of development oforthodox seeds there is the reduction in the volumeof water-filled vacuoles, the de-differentiation oforganelles and the shut down of metabolism thatare necessary for desiccation tolerance. However,other processes, involving particularly LateEmbryogenic Abundant (LEA) proteins andsugars, also occur. LEA proteins accumulateduring the later stages of embryogenesis inorthodox seeds (Galau et al., 1986, 1991).

These proteins are similar to the

dehydrins produced on drying of vegetativetissue. In fact, dehydrins are a subset of LEAs.These proteins are thought to have a

protective role in the dry state, or certainly tocontribute in some way to desiccation tolerance(reviewed by Oliver and Bewley, 1997;Kermode, 1997), although details of their modeof action are not yet clear.

LEAs have been shown to be present inquite a wide range of recalcitrant seeds, mostlytemperate (Bradford and Chandler, 1992; Finch-

Savage et al., 1994a), but also some tropicalspecies (Gee et al., 1994; Farrant et al., 1996).However, they are not universally present in allrecalcitrant seeds; they are certainly absent fromthe axes of several tropical wetland species(Farrant et al., 1996). It is possible that thepresence of LEAs can contribute to the slightlygreater desiccation tolerance and chilling toleranceof some recalcitrant species.

If sucrose and oligosaccharides such asraffinose are present in the correct ratios, then a

glass can be formed as water is lost. This state ishighly viscous, which should slow down anychemical processes occurring in the seed, thusproviding protection on desiccation (Williams andLeopold, 1989; Leopold et al., 1994). Sugars,possibly in the appropriate mass ratios, occurin many recalcitrant seeds (Farrant et al., 1993;

Finch-Savage and Blake, 1994; Lin and Huang,1994; Steadman et al., 1996), and it is possible thatglasses may form on dehydration. However, these

glasses, if they do form, are obviously not effectiveprotectants, as dehydration does kill recalcitrantseeds.

To summarise, some species ofrecalcitrant seeds do have the appropriate sugarsand some have dehydrins. However, it should bepointed out that these mechanisms are thought tooperate and to provide protection at low watercontents. On dehydration recalcitrant seeds die atwater contents higher than those at which theseprotective measures are thought to operate.Whatever the role of LEAs and sugars may be inorthodox seeds, they are not pertinent torecalcitrant seeds.

It is now relatively well established thattreatments which disturb normal metabolism cangenerate free radicals, and protection againstthese free radicals by anti-oxidant systems isessential for survival. Highly active anti-oxidantsystems are certainly very important in theprotection mechanisms of resurrection plants,both on drying and during rehydration (Oliverand Bewley, 1997). Although not a lot of workhas been done, these mechanisms are present inorthodox seeds.

During the drying of recalcitrant seeds,stable free radicals do accumulate (Hendry et al.,1992; Finch-Savage et al., 1993, 1994b). There isevidence that anti-oxidant systems also fail(Hendry et al., 1992; Finch-Savage et al., 1993), orthey may not be present at sufficient levels.Oxidative damage probably does occur duringdrying.

There have been some new suggestionsconcerning the mechanisms of desiccationtolerance (Hoekstra et al., 1997; Golovina et al.,1998). These authors postulate the existence ofamphipathic molecules that become integrated intothe membrane as water is lost. It is suggested thatthese molecules lower the water content at which


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lipid phase changes occur and thus possiblystabilize the integral proteins in the membranes.This is recent work and so far no information is

available with regard to the presence of thesemolecules in recalcitrant seeds.

It is apparent that there is a suite, acollection, of mechanisms that are thought toprotect against desiccation damage. Some of theseprotective mechanisms probably also operateduring rehydration, as well as during drying. Thedifferent protective mechanisms seem to operate indifferent water content ranges. All thesemechanisms must be operational for desiccationtolerance. Recalcitrant seeds lack some or all ofthese mechanisms. It is not apparent whether thereis any relationship between the degree ofrecalcitrance of a seed and the extent to which ithas or expresses some of these mechanisms.

THE IMPORTANCE OF DEHYDRATION

RATE

Dehydration rate is perhaps one of themost confusing issues in recalcitrant seedphysiology. When whole seeds are dried relativelyslowly - over several days - viability is generallylost at embryonic axis water contents in the rangeof 1.0 - 0.5 g water/g dry mass (50 - 35% wet massbasis). However, if isolated embryonic axes aredried rapidly (minutes to hours) they survive tomuch lower water contents - 0.45 to 0.25 g/g - andpossibly even lower (e.g. Pammenter et al., 1991).Thus the effect of dehydration depends upon therate of dehydration. This makes it extremelydifficult to assess or measure desiccation tolerance

- the response depends upon how the tissue isdried. However, this approach could be criticisedbecause of the experimental protocol; the effectcould be due to different drying rates, or it couldbe due to the fact that different tissues were used toachieve the different drying rates (embryonic axesfor rapid drying and whole seeds for slow drying);

thus for rapid drying the embryonic axis has beenremoved 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 producesseeds about the size of a peanut with a hardendocarp. If the endocarp is removed the seedscan be dried by burying in silica gel to an axiswater content of around 0.2 g/g (dry mass basis)in about 48 h (Figure 2a). If the endocarp is leftintact it takes about 10 d for axis water contentto reduce to 0.5 g/g (Figure 2b). We alsomanipulated the drying technique toachieve what we describe as an intermediatedrying rate.

The response of final germination towater content using the three drying treatments isseen in Figure 3. Seeds that were dried slowlystarted to lose viability at an axis water content ofabout 1.3 g/g and were all dead at 0.6 g/g. Seedsthat were dried rapidly retained 90% viability to awater content of 0.5 g/g, and were all dead at 0.2g/g. Seeds dried at an intermediate rate showed anintermediate response to desiccation. The rate ofdrying does have a marked effect on the watercontent tolerated; the effect is not one of removingthe axis from the seed. Why should this be thecase?

The cells of embryonic axes of the seedsdried at different rates showed very differentultrastructural responses to dehydration (see Berjakand Pammenter, this volume). In seeds that weredried slowly to the extent that considerableviability loss occurred, there was advanceddegradation of membrane structures, particularly

in the plastids, and an abnormal appearance ofthe lipid bodies, particularly a darkening of thesurface. Seeds that had been dried rapidly to aslightly lower water content, but still were mostlyviable, generally showed well preservedmembranes and nuclei. Although membranedamage did ultimately occur on rapid drying, there


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was very marked degradation at high watercontents in slowly dried material. Thisultrastructural evidence suggests that different

deteriorative processes may be occurring at thedifferent drying rates.

To understand the effect of drying ratewe have to appreciate that some damagingprocesses are aqueous based. Slowly driedmaterial will spend a long time at theintermediate water contents where damage from

these aqueous based processes can accumulate.However, if material is dried rapidly, only ashort time is spent at these intermediate water

contents, so little damage accumulates. Thus thefaster the drying, the less damage thataccumulates and the lower the water content thatcan be tolerated. However, there is a lower limit- no matter how fast recalcitrant seeds are dried,they cannot tolerate the low water contentstypical of orthodox seeds.

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

0,0

0,5

1,0

1,5

2,0

0 20 40 60 80

Drying time (h)

Axiswatercontent(g/g)

a

0,0

0,5

1,0

1,5

2,0

2,5

0 2 4 6 8 10

Drying time (d)

Axiswatercontent(g/g)

slow

intermediate

b


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FIGURE 3 - Final germination of seeds of Ekebergia capensisdried at different rates to a range of axiswater contents.

We visualise two types of damage thatcan occur during drying. As a consequence ofdisturbed metabolism, free radical-mediatedoxidation processes can proceed. These reactions areaqueous-based and occur at intermediate watercontents. The protection mechanisms against thistype of damage are the switching-off of metabolism,de-differentiation of organelles, and efficient anti-oxidant systems. The second type of damage we

envisage is due to the biophysical disruptions causedby the removal of water from macromolecular andmembrane surfaces. These occur at low watercontents. Protection mechanisms against this type ofdamage include LEAs, vitrification by sugars,and possibly the amphipathic moleculessuggested to migrate between cytosol andmembranes on dehydration and rehydration.

EVOLUTIONARY STATUS OF

RECALCITRANT SEEDS

In vegetative tissue desiccationsensitivity is probably the ancestral state, buttolerance evolved independently a number oftimes, including more than once in theangiosperms (Oliver and Bewley, 1997). Anyattempt at a similar phylogenetic analysis using

seed desiccation response as a character state fails -both tolerance and sensitivity occur in all majorclades of extant seed plants. However, it isprobable that sensitivity is the ancestral state, buttolerance evolved independently a number of times(von Teichman and van Wyk, 1994; Pammenter andBerjak, 2000). This raises the interesting question asto whether the mechanisms of tolerance are

necessarily the same in all seed species.

ECOLOGICAL CONSIDERATIONS

A characteristic of recalcitrant seeds istheir limited lifespan, and this will placeconstraints on the range of environmentalconditions in which regeneration via seeds canoccur. However, when we try to get detailedinformation on the seed ecology of speciesproducing recalcitrant seeds, we have a major

problem in the past ecologists and seed scientistshave not often communicated. Ecologists study thecomposition of soil seed banks, the lifespan ofseeds in these banks, and environmental conditionsinitiating germination, but often have noinformation as to the water content of the seedsof interest. Seed physiologists, on the other

0

20

40

60

80

100

0,0 0,5 1,0 1,5 2,0 2,5

Axis water content (g/g)

Germination(%)

rapid

slow

intermediate


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hand, have a good knowledge of only a fewspecies, mostly economically important ones(either crop species or weedy invaders), and only

occasionally become involved in ecologicalconsiderations.

Recalcitrant seeds are common in mesictropical forests. These conditions generally wouldbe favourable for germination and seedlingestablishment, and so there has been no pressuredriving the evolution of desiccation tolerance, orthe characteristic has been secondarily lost. Seedecologists will tell us that seeds of climax speciesof these ecosystems are not often found in the soilseed bank their lifespans are low, whereas seedsof gap specialists are well represented in the soilseed bank.

The seeds of climax species generallyshow no dormancy, germinate rapidly (unless theyhave a hard covering), are absent from the soil seedbank and these species persist as a seedling bank.They are generally large and not wind dispersed.These are the characteristics that one would expectfrom recalcitrant seeds, and it is highly probablethat most of these seed species are indeedrecalcitrant, 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 meansconfined to these systems. Species producingrecalcitrant seeds do occur in more seasonalhabitats, and a number of strategies have evolvedto permit successful seedling establishment. Manytemperate recalcitrant seeds are shed in autumnand over-winter as seeds. A necessary prerequisitefor this, of course, is chilling tolerance. Because ofthe low temperatures, rates of germination will be

slow, and will not be completed before the arrivalof spring. In fact some species such as Aesculushippocastanum actually have a chillingrequirement for germination (Pritchard et al.,1996). Low temperatures and general dampnesswill also slow water loss, reducing the risk of deathby desiccation. As a generalisation, recalcitrant

species of temperate regions are generally moredesiccation tolerant and have longer life spans thanthose of tropical origin.

REFERENCES

BERJAK, P.; PAMMENTER, N.W. &VERTUCCI, C.W. Homoiohydrous(recalcitrant) seeds: developmental status,desiccation sensitivity and the state of water inaxes of Landolphia kirkii Dyer. Planta,186:249-261,1992.

BERJAK, P.; VERTUCCI, C.W. & PAMMENTER,N W. Effects of developmental status anddehydration rate on characteristics of waterand desiccation-sensitivity in recalcitrant seeds ofCamellia sinensis. Seed Science Research,3:155-166, 1993.

BRADFORD, K.J. & CHANDLER, P.M.Expression of dehydrin-like proteins inembryos and seedlings ofZizania palustrisandOryza sativa during dehydration. PlantPhysiology,99:488-494, 1992.

FARRANT, J.M.; BERJAK, P. & PAMMENTER,N.W. The effect of drying rate on viabilityretention of recalcitrant propagules of

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FARRANT, J.M.; PAMMENTER, N.W. &BERJAK, P. The increasing desiccationsensitivity of recalcitrant Avicennia marina

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FARRANT, J.M.; PAMMENTER, N.W. &BERJAK, P. Recalcitrance - a currentassessment. Seed Science and Technology,16:155-166, 1988.


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FARRANT, J.M.; PAMMENTER, N.W. &BERJAK, P. Germination-associated eventsand the desiccation sensitivity of recalcitrant

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FARRANT, J.M.; PAMMENTER, N.W.;BERJAK, P.; FARNSWORTH, E.J. &VERTUCCI, C.W. Presence of dehydrin-likeproteins and levels of abscisic acid inrecalcitrant (desiccation sensitive) seeds maybe related to habitat. Seed Science Research,6:175-182, 1996.

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FINCH-SAVAGE, W.E. Seed water status andsurvival in the recalcitrant species Quercusrobur L.: Evidence for a critical moisturecontent. Journal of Experimental Botany,43:671-679, 1992.

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HENDRY, G.A.F. & ATHERTON, N.M.Embryo water status and loss of viabilityduring desiccation in the recalcitrant seedspecies Quercus robur L. In: CME, D. &CORBINEAU, F. (Eds.) Fourth internationalworkshop on seeds: Basic and appliedaspects of seed biology.Paris, ASFIS, 1993. p.723-730.

FINCH-SAVAGE, W.E.; HENDRY, G.A.F. &ATHERTON, N.M. Free radical activity andloss of viability during drying of desiccation-

sensitive tree seeds. Proceedings of theRoyal Society of Edinburgh, 102B:257-260, 1994b.

FINCH-SAVAGE, W.E.; PRAMANIK, S.K. &BEWLEY, J.D. The expression of dehydrinproteins in desiccation-sensitive (recalcitrant)seeds of temperate trees. Planta, 193:478-485,1994a.

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