South African Journal of Botany 72 (2006) 167 – 176 www.elsevier.com/locate/sajb

Viability and ultrastructural responses of seeds and embryonic axes of emetica to different dehydration and storage conditions

J.I. Kioko *, P. Berjak, N.W. Pammenter

School of Biological and Conservation Sciences, University of KwaZulu-Natal, Durban 4041, South Africa

Received 16 June 2005; accepted 1 July 2005

Abstract

The seeds of Trichilia emetica, a multi-purpose tropical forest species, displayed typical recalcitrant behaviour, being shed at an average axis water concentration of 2.82 g per g dry matter (g gÀ 1), and losing viability when dehydrated to axis water concentrations below 0.42 and 0.26 g gÀ 1, when dried slowly or rapidly, respectively. The ultrastructure at shedding was indicative of active metabolism, as would be expected of mature recalcitrant seeds which grade into germinative metabolism after shedding. Rapid dehydration enabled the maintenance of ultrastructural integrity to water concentrations as low as 0.3 g gÀ 1, while cells of axes dried slowly to similar water concentrations displayed total subcellular destruction. In the fully hydrated state, the storage lifespan of the seeds was limited to 60 days at 16 -C, after which all the seeds had germinated in storage. Ultrastructural examination, however, indicated that prolonged mild water stress had occurred, which the seeds are suggested to have suffered as germination proceeded in storage. When stored at 6 -C, the seeds showed extensive ultrastructural derangement, which was accompanied by loss of viability after 20 days, presumably as a result of chilling injury, while storage at 25 -C resulted in all seeds germinating in storage in 35–40 days. Even though the seedcoat has been shown to inhibit germination, it did not appear to affect seed longevity or germination in storage at any of the temperatures used. D 2005 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Desiccation sensitivity; Medicinal ; Recalcitrant seed; Seed storage; Trichilia emetica

1. Introduction species has a wide range of uses, including the use of the bark in herbal medicine, the use of oil from the seeds as food and for Trichilia emetica Vahl. typifies many tropical species cosmetic purposes, and the provision of wood products (Watt producing recalcitrant seeds: a high exploitation and extinction and Breyer-Brandwijk, 1962; Mabogo, 1990; Pooley, 1993). rate (UNEP, 1995; Scott-Shaw, 1999; Pitman et al., 2002), The species is not considered threatened, but the available intensified by the absence of any, or at least useful, information populations in South Africa may be only those planted in cities, on the physiology and/or storability of the seeds. This lack of with no wild populations encountered in the course of this study. information thwarts efforts aimed at conservation, considering Seed production by T. emetica is often abundant, but mature that seeds provide the safest and the least costly means of seeds are available for a relatively short period in the seeding germplasm conservation and are therefore widely utilised in, season (usually c. 3 weeks, according to our observations), and and form the basis of, the establishment of seed-banks and the seeds are reported to have short longevity and are gene-banks around the world. Successful seed storage, recommended to be sown within three days of collection however, requires an understanding and prudent exploitation (Hines and Eckman, 1993). Within this period, the seeds are of the post-shedding physiology of the seeds. overrun by fungi unless the aril is removed and the seed T. emetica grows in forests along the eastern coast of southern surface-sterilized, a treatment that, however, delays fungal Africa and in open riverine-alluvial lowland rainforests of contamination only by a matter of days (Myeza, 2005). tropical Africa (Beentje, 1994). Throughout its range, the This investigation sought to establish the response of the seeds and/or embryonic axes of T. emetica to various * Corresponding author. dehydration and storage regimes, with the aim of understand- E-mail address: [email protected] (J.I. Kioko). ing the post-harvest physiology of the seeds and establishing

0254-6299/$ - see front matter D 2005 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2005.07.001 168 J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176 methods for short- to medium-term storage of the zygotic rehydration if dried) by soaking in 0.2% (w/v) mercuric germplasm. This will also contribute to the slight body of chloride for 1min, followed by washing with sterile distilled knowledge on the seed behaviour of tropical species. water (three changes), and then cultured aseptically on an MS (Murashige and Skoog, 1962) nutrient medium supplemented 2. Materials and methods with 3% sucrose and solidified with 0.8% agar, and placed in a growth-room using a photoperiod of 16 h light, 8 h dark at 2.1. Seed procurement and handling temperatures of 25 and 18 -C, respectively. Axes were scored as having germinated on the basis of radicle elongation beyond Mature, newly opened fruits were harvested directly from 5 mm, while shoot development was determined by the trees growing at La Lucia, KwaZulu-Natal (29-45S,V 31-4E)V appearance of the first pair of leaves. Embryonic axes which and the seeds extracted immediately and processed for produced callus instead of either roots or shoots were scored as experiments within 48 h. Seeds destined for storage without viable but not germinated. the testae were de-coated manually, while those to be stored intact were soaked in water for 3 h to facilitate removal of the 2.6. Ultrastructural studies aril. Cleaned seeds were then surface-sterilized by soaking in 1% sodium hypochlorite solution for 20 min, air-dried back to Small pieces of root and/or shoot apices, not exceeding the original fresh weight, and dusted with a benomyl fungicide. 0.5 mm3 in size, were cut from five replicate embryonic axes and immediately fixed, or, if dehydrated, rehydrated for 30 2.2. Seed storage min, and then processed for transmission electron microscopy following glutaraldehyde-osmium fixation (Wesley-Smith, Following cleaning and sterilization, seeds were stored in a 2001). monolayer on plastic mesh pre-sterilized by soaking in 1% sodium hypochlorite for 20 min. This mesh was supported 2.7. Electrolyte conductivity about 100 mm over water-saturated paper towelling enclosed in 5-l translucent plastic buckets which were sealed and placed at The leakage of electrolytes from embryonic axes was 6, 16, or 25 -C. measured using a CM100\ multi-cell conductivity meter (Reid and Associates, Durban, South Africa). The conductiv- 2.3. Determination of water concentration ities of the leachate from ten replicates of individual axes from each treatment (or axes excised from seeds following treat- Water concentration of the different seed components was ment), each soaked immediately after dehydration in 3 ml determined on individual seeds and axes gravimetrically. The deionised water, were measured every 30 min for 18 h. Rate of material was heated in an oven at 80 -C for 48 h to constant leakage was expressed in microsiemens per centimeter per weight, and the water concentration expressed as gram water gram dry matter per minute. per gram dry matter (g gÀ 1). 2.8. Sampling 2.4. Drying At each sampling interval, 40 seeds and/or 40 embryonic Two rates of drying were employed. Slow drying was axes were randomly sampled, and used as follows: 20 for carried out by maintaining whole seeds on a plastic mesh viability assessment, 10 for electrolyte conductivity, five for placed 200 mm above activated silica gel in a closed 20-l plastic water concentration determination, and five for ultrastructural bucket, while fast drying (performed on embryonic axes only, studies. as the seeds are too large to dry sufficiently rapidly) was achieved by flash-drying (Berjak et al., 1990; Pammenter et al., 3. Results and discussions 2002) for periods of up to 120 min. 3.1. The effect of dehydration on the viability and ultrastruc- 2.5. Viability assessment ture of seeds and embryonic axes

Whole seeds were sown in moist vermiculite (buried just The direct relationship between the rate of dehydration and deep enough to cover the seed), and maintained in a the degree of desiccation tolerated by recalcitrant seed tissues germination room (16 h light, 8 h dark) at 25 -C, germination has been unequivocally demonstrated (reviewed by Pammenter being scored daily. A seed was scored as having germinated on and Berjak, 1999; Berjak and Pammenter, 2001). For large the basis of radicle protrusion. For each sample, percentage seeds, rapid rates of drying are generally unattainable simply germination and the germination rate (the maximum percentage because of their size, which necessitates the use of excised germination attained divided by the number of days taken to embryonic axes in operations that require rapid decrease in attain that percentage) were determined. water concentration. The mean mass of T. emetica seeds used The viability of excised embryonic axes was determined by in this study was 0.36T0.10 g, while that of excised embryonic in vitro germination. Axes were surface-sterilized (after axes was 0.0013T0.0004 g (average massTs.d. of 10 randomly J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176 169 chosen individual seeds and/or embryonic axes). Thus, while 100 embryonic axes dried to nearly constant water concentrations within 120 min, whole seeds approached similar water 80 concentrations only after 5 days of drying (Fig. 1). The average water concentration of axes newly excised from 60 fresh seeds was 2.82T0.50 g gÀ 1 (averageTs.d.), and these had 100% germination, as did whole seeds. There was a reduction 40 in viability with increasing degree of dehydration, with slowly 20 dried seeds being ungerminable at axis water concentrations of À 1 0.42 g g (attained after 3 days). However, 85% of excised 0 embryonic axes retained viability at a similar water concentra- % germination / shoot formation 0 0.2 0.4 0.6 0.8 1 1.2 1.4 tion following flash-drying for 60 min, and an essentially Water concentration (g/g) similar percentage viability was maintained to considerably À 1 Fig. 2. The effect of water concentration on germination of slowly dried T. lower water concentrations (c. 0.3 g g ) after flash-drying for emetica seeds (n), flash-dried embryonic axes (>), and on shoot formation in 90 min (Fig. 2). Non-dehydrated embryonic axes of T. emetica flash-dried embryonic axes (q). had an ultrastructure typical of freshly harvested recalcitrant seeds, with features indicative of ongoing active metabolism such as well-developed mitochondria, profiles of rough Flash-drying for 30 min achieved a similar axis water endoplasmic reticulum, polysomes, Golgi bodies, and a limited concentration, about 0.5 g gÀ 1, as did slow drying for three degree of vacuolation (Figs. 3a,b and 4a,b). Chloroplasts days (Fig. 1), but the ultrastructural situation in the axis cells showing considerable inner membrane development were a of the material dried at the two different rates was markedly feature of the shoot meristems (Fig. 4b), and the cytomatrical different: cells from the flash-dried axes had retained organisation in the non-dried condition was such that orga- cytomatrical organisation (Fig. 3c), with well-developed nelles were normally distributed throughout the cell (Figs. 3a mitochondria, many, relatively short profiles of rough and 4a), an indication of the underlying cytoskeletal organisa- endoplasmic reticulum, Golgi bodies, and polysomes (Fig. tion (Berjak et al., 1999). 3d), indicating the metabolic potential of the axes. In stark contrast, cells from axes of seeds dehydrated to similar water concentrations (c. 0.5 g gÀ 1) over three days displayed total 3.5 a subcellular destruction (Fig. 3e). The plasmalemma was 3 completely detached from the cell wall and the entire cell 2.5 contents had coalesced to such an extent that it was impossible to discern individual organelles. Similar ultra- 2 structural deterioration in flash-dried axes was observed only 1.5 when they had been dehydrated to about half that water concentration, 0.26 g gÀ 1 (Fig. 3f). 1 In axes flash-dried for 90 min to a water concentration of À 1 0.5 about0.3gg (80% germination and 25% shoot water concentration (g/g) formation), cells from root meristems presented an ultra- 0 structure consistent with normal functioning including 0 153045607590105120 cristate mitochondria, discrete Golgi bodies, endoplasmic Flash-drying time (min) reticulum profiles, and well-defined nuclear and vacuolar 4 membranes (Fig. 4c,d). However, many cells from the shoot b 3.5 meristem exhibited distorted organelles with occasional 3 blistering (Fig. 4e,f). As shown in Fig. 6,axiswater concentrations of 0.3 g gÀ 1 are not representative of those 2.5 of the shoot-pole, which dries considerably more. Hence, in 2 the shoot meristem (which occupies the most superficial 1.5 position and hence is least protected), water concentrations may have been at damaging levels for most of the 90-min 1 dehydration period, thus accounting for the marked ultra- Water concentration (g/g) 0.5 structural abnormality present. 0 Besides ultrastructural and viability characteristics, another 0123456 measure of desiccation damage in seeds is the loss of cell Dehydration period (days) membrane integrity, which can be expressed in terms of the Fig. 1. Drying time course for T. emetica axes dehydrated after excision (a) or rate and extent of electrolyte leakage from the tissues. This within the seed (b). Markers represent meanTstandard deviation of five has been used successfully to assess dehydration damage for individual axes. desiccation-sensitive seed tissues of a variety of species (e.g. 170 J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176

Fig. 3. The ultrastructural status of root meristematic cells of T. emetica in the newly harvested, non-dehydrated state (a and b), and, following brief rehydration, after flash-drying (c and d) or slow drying (e) to c. 0.5 g gÀ 1. The effects of excessive dehydration are shown in (f). (a) The cells were typified by slightly irregular nuclear profiles and a few well-developed vacuoles, with organelles distributed throughout the cytomatrix. (b) Well-developed organelles, elements of the endomembrane system, and polysomes were indicative of active metabolism. (c) After flash-drying to c. 0.5 g gÀ 1 over 30 min, the intracellular integrity was generally maintained. (d) The occurrence of many cristate mitochondria, Golgi bodies (not illustrated), and endoplasmic reticulum was indicative that the potential for metabolic activity in the axes was not curtailed by the flash-drying. (e) When the axes were dehydrated over 3 days to a similar water concentration as those illustrated in (c) and (d), the ultrastructure was completely deranged, and no organelles could be discerned. (f) Flash-drying to a water concentration of c. 0.26 g gÀ 1 was excessive, causing desiccation damage sensu stricto (e). CW, cell wall; er, endoplasmic reticulum; Gb, Golgi body; m, mitochondrion; N, nucleus; Nu, nucleolus; ne, nuclear envelope; p, plastid; v, vacuole. Bar=500 nm.

Vertucci and Leopold, 1987; Li and Sun, 1999; Wesley- maintenance of the integrity of cellular membranes during Smith et al., 2001). In the present study, the rate of dehydration and subsequent rehydration. electrolyte leakage was observed to increase with decreasing Rapid dehydration of desiccation-sensitive seed tissues water concentration in embryonic axes, with the extent of facilitates viability retention to lower water concentrations than leakage being greater in axes dried slowly within whole would be tolerated by slow drying. This is presumed to be the seeds (Fig. 5). A similar relationship between drying rate and outcome of the limited time afforded (during drying), in which electrolyte leakage was reported for embryonic axes of deleterious metabolism-linked damage can occur (Pammenter (Artocarpus heterophyllus)byWesley-Smith et al. and Berjak, 1999; Walters et al., 2001). Damage to subcellular (2001), and indicates that flash-drying contributes to the membranes — which is curtailed during rapid drying — is J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176 171

Fig. 4. (a) Cells from the shoot meristem contained a number of smallish vacuoles, showing evidence of autophagic activity (arrow-head), and nuclei of slightly irregular profile, containing prominent nucleoli. (b) A generally even distribution of organelles, elements of the endomembrane, and membrane-associated and cytomatrical polysomes (arrow-head) was typical. (c and d) After dehydration c. 0.3 g gÀ 1, over 90 min, cells from the root meristem maintained the structure consistent with normal functional cells. (e and f) In contrast cells from the shoot meristems had abnormally shaped organelles (arrow-heads, e), with irregularities such as the blistered membrane boundary of the plastid illustrated in (f), arrowed CW, cell wall; er, endoplasmic reticulum; Gb, Golgi body; m, mitochondrion; N, nucleus; Nu, nucleolus; ne, nuclear envelope; ch, chloroplast. Bar=500 nm. prominent among the deleterious consequences of slow dehy- Regardless of the rate at which recalcitrant embryonic axes dration (Kioko et al., 1998; Pammenter et al., 1998). According are desiccated, however, there is a lower water concentration to Berjak et al. (1989), slow drying — certainly over a period of limit below which viability will be rapidly lost (Pammenter and days, as was done in this study — allows the seeds to progress Berjak, 1999), and this limit is higher than that tolerated for along the germination pathway which is accompanied by their extended periods by orthodox seeds, which can be desiccated increasing desiccation-sensitivity, while keeping the tissues at to water concentrations 0.05 g gÀ 1 without losing viability Fintermediate_ water concentrations at which active, regulated (Hong and Ellis, 1996). A spectrum of factors is implicated in metabolism gives way to de-regulated, Fout-of-phase_ metabo- the loss of viability as a result of desiccation in recalcitrant lism (Pammenter et al., 1998) and generation of free radicals, seeds, among which is their inability (unlike orthodox seeds) to which, in turn, cause peroxidative membrane damage (Leprince tolerate the loss of non-freezable water (Pammenter et al., et al., 1990; Smith and Berjak, 1995). 1991). These authors, working on the embryonic axes of 172 J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176

80 techniques such as cryopreservation, because abnormal growth 70 results after thawing/rehydration (Normah and Marzalina,

) 60 1996; Kioko et al., 1998; Wesley-Smith et al., 2001). Efforts -1

g were therefore made, in this study, to achieve a more uniform

-1 50 drying rate throughout individual embryonic axes, by encap-

min 40 sulating the axis shoot-tips with an alginate gel (Motete et al., -1 30 1997) before desiccation. Such encapsulation has been S cm

µ suggested to have a possible advantage in terms of reduction

( 20 of freezing injury during cryopreservation (Dereuddre et al., 10

Rate of electrolyte leakage 1990). Encapsulation, however, did not substantially alter the 0 pattern of drying in the embryonic axes, and the shoot-tip still 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 dried faster than the root apex (Fig. 6b). It therefore appears water concentration (g/g) that the putative protection afforded to the root meristem by the Fig. 5. The rate of electrolyte leakage from embryonic axes of T. emetica root-cap during dehydration cannot be mimicked by an alginate dehydrated either slowly (dotted line, r) or rapidly (solid line, g). Curves were layer, the hydrodynamics of which would be different from that fitted with Microsoft Excel 2000. in a living tissue.

Landolphia kirkii, suggested that the amount of non-freezable 3.2. The effect of dehydration on the rate of germination water required to maintain intracellular integrity represents the Fminimum tolerance level_. Finch-Savage (1992) also identi- Stimulation of germination by short-term non-injurious fied Fcritical moisture concentrations_ in Quercus robur, equal dehydration may be a characteristic of recalcitrant seeds to the amount of Fmatric-bound water_, below which those (Pammenter and Berjak, 1999), and has been observed in other seeds could not withstand dehydration. However, subsequent species studied, e.g. Ekebergia capensis (Pammenter et al., studies by Pammenter et al. (1993) demonstrated that 1998), although the basis for such stimulation is only now being sensitivity to very rapid desiccation in recalcitrant seeds cannot investigated. This effect was not apparent for T. emetica under be explained in terms of structure-associated water alone, as the conditions of the present experiments (Fig. 7). It is, however, (those authors showed) axes of some species lose viability at possible that germination in seeds of T. emetica may be water concentrations in excess of the bound water levels in their tissues. 2 It was noteworthy, in this study, that flash-dried embryonic a axes lost the capacity for shoot development at water concentra- 1.5 tions at which germination (as defined by radicle growth) still took place (Fig. 2). This is not uncommon in studies dealing with dehydration of embryonic axes of recalcitrant seeds, and 1 survival is frequently reported only in terms of root growth (e.g. Theobroma cacao [Chandel et al., 1995] and Araucaria 0.5 hunstenii [Pritchard and Prendergast, 1986]). In more extreme cases, shoot formation has been observed only in control, non- water concentration (g/g) 0 dried axes, with all dehydration treatments precluding the ability 0 30 60 90 120 of the axes for shoot formation (e.g. Artocarpus heterophyllus drying time (min) [Wesley-Smith et al., 2001]). The differential response of shoot- and root-tips to 3 b dehydration is likely to be a consequence of different drying 2.5 rates at the two poles of individual embryonic axes, resulting in different shoot and root water concentrations in the extremities 2 of individual axes by the end of each drying period. As shown in Fig. 6a, the water concentration of the shoot-pole was higher 1.5 than that of the root-pole in freshly harvested material. Upon 1 dehydration, however, the shoots of individual axes initially dried more rapidly than the roots, after which the root and 0.5 water concentration (g/g) shoot water concentrations declined in parallel, with that of the 0 shoots being lower than of the roots. The result of this drying 0 30 60 90 120 pattern is suggested to have imposed a more severe stress upon drying time (min) the shoot meristem for a longer period. Fig. 6. Water concentrations of shoot-tips (D) and root-tips (>) of axes of T. Differential drying within embryonic axes could limit the emetica flash-dried either naked (a) or encapsulated in sodium alginate (b). possibility of desiccating these explants to water concentrations Markers represent the averageTstandard deviation of five individual embryonic low enough to facilitate the application of conservation axes. J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176 173

100 stored at 6 -C, in addition to 16 and 25 -C. Those seeds stored at 6 -C steadily lost germinability with storage time, 80 with total loss of viability after 20 and 25 days (when stored without or with testae, respectively), as illustrated in Fig. 8a and b. On the other hand, there was no apparent 60 change in viability for seeds stored either at 16 or 25 -C. At 16 -C, the seeds stored with or without testae maintained 40 almost total germinability for 60 days, although all had % germination germinated in storage by then. Seeds stored at 25 -C 20 attained total germination in storage within 35 and 40 days (with testae removed and intact, respectively). The germi- - 0 nation rate declined steadily in all seeds stored at 6 C, and 0 5 10 15 20 increased steadily in all seeds stored at either 16 or 25 -C days after sowing (Fig. 8c and d). It was notable that there was little discernible fungal Fig. 7. Germination time course of T. emetica seeds dehydrated over silica gel to - different axis water concentrations: >, not dried (2.8 g gÀ 1); D, dried to 1.2 g gÀ 1 proliferation on seeds stored either at 16 or 25 C, while those over 1 day; n, dried to 0.7 g gÀ 1 over 2 days; g, dried to 0.42 g gÀ 1 over 3 days. stored at 6 -C were overrun by fungi and bacteria within 20 The slope of each germination curve represents the germination rate. days. As all the seeds were from a common batch, this strongly suggests that vigorous, metabolically active recalcitrant seeds stimulated by a dehydration interval(s) shorter than 1 day, as have mechanisms to counteract micro-organism proliferation, water concentration was presently more than halved during this as demonstrated by Anguelova-Merhar et al. (2003) and period. Calistru (2004). Ultrastructural studies showed that, while freshly har- 3.3. Storage longevity vested seeds were in an actively metabolic state (Figs. 3a,b and 4a,b), those stored at 6 -C showed signs of deterioration In an attempt to curtail metabolic rate and prolong the by day 10: for example, the cells had electron-translucent time before germination in storage occurred, the seeds were mitochondria and plastids with no discernible internal

100 100

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40 40 % germination % germination 20 20 a b 0 0 0 102030405060 0 102030405060 Storage period (days) Storage period (days)

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10 10 Germination rate Germination index (% germination/day) (% germination/day) 5 c 5 d 0 0 0 102030405060 0 102030405060 Storage period (days) Storage period (days)

Fig. 8. (a –b): Final percentage germination of T. emetica seeds stored with testae (a) or without testae (b), at 6 -C(>), 16 -C(r) and 25 -C(g). All seeds stored at 16 and 25 -C had germinated in storage by day 60. (c–d): The germination rate after various storage intervals, calculated as the maximum germination attained divided by the number of days taken to reach it, of T. emetica seeds stored with testae (c) or without (d), at 6 -C(>), 16 -C(r) and 25 -C(g). 174 J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176 structure (not illustrated). By day 20 of storage at 6 -C, the and coordination, which occurs when enzymes are cold-labile cells exhibited profound ultrastructural derangement: the (Guy, 1990; Salahas et al., 2002) or when lipids of the plasmalemma had receded from the cell wall and was mitochondrial inner membranes change from the liquid discontinuous in many places, and organelles were exten- crystalline to the gel state (Bedi and Basra, 1993; Berjak sively degraded (Fig. 9a). This damage is suggested to be et al., 1995). Further derangement of metabolism would the result of chilling injury to which many tropical result from the dismantling of cytoskelatal elements, which recalcitrant seeds are thought to be susceptible (Chin and has been shown to occur at low temperatures in chilling- Roberts, 1980). sensitive plant tissues (Raison and Orr, 1990), as certain The basis of chilling injury in seeds is not clear, but is biochemical pathways operate as multi-enzyme complexes suggested to include the impairment of respiratory capacity anchored to the cytoskeleton (Masters, 1984, 1996). Sus-

Fig. 9. The ultrastructure of root meristematic cells of seeds of T. emetica stored at 6, 16, and 25 -C for various periods. (a) After 20 days in storage at 6 -C, there was extensive ultrastructural damage, including conspicuous withdrawal of the plasmalemma (arrow-head) from the cell wall. In contrast, cells from seeds maintained at 16 -C (b and c) and 25 -C (d and e) presented evidence of ongoing metabolic activity, with well-developed mitochondria and plastids, some of which contained substantial starch grains, rough endoplasmic reticulum and polysomes, as well as Golgi bodies. (f) After 60 days in storage at 16 -C cells had characteristically undulating cell walls (arrow-heads), electron-translucent mitochondria with poorly defined cristae, and vesicles and tubular structures presumably formed by degraded plasma membrane were evident (arrow-heads in inset). CW, cell wall; er, endoplasmic reticulum; Gb, Golgi body; m, mitochondrion; N, nucleus; Nu, nucleolus; pm, plasma membrane. Bar=500 nm. J.I. Kioko et al. / South African Journal of Botany 72 (2006) 167–176 175

3.5 increase the storage longevity of the seeds (e.g. Motete et al., 3 1997). While seed storage fungi have been shown to have a central 2.5 role in the viability loss of stored recalcitrant seeds (e.g. Calistru et al., 2000; Berjak et al., 2004), fungal proliferation 2 was evident in only one or two seeds out of 30 at each sampling interval, even after 60 days of storage at 16 -C (or 1.5 after 40 days at 25 -C). However, the ultimately deteriorating 1 ultrastructural state, coupled with the germination of all seeds in storage, indicates that seeds of T. emetica have a useful water concentration (g/g) 0.5 storage lifespan of not much longer than two months at cool (c. 16 -C) temperatures. The long-term preservation of the zygotic 0 0 102030405060 germplasm of this, and other recalcitrant-seeded species, may days after storage be achieved only through the cryopreservation of excised embryonic axes. Fig. 10. The water concentrations of axes (solid lines) and cotyledons (dashed lines), over time in storage time, of testa-enclosed T. emetica seeds at 4 -C(>), - r - g 16 C( ) and 25 C( ). References

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