South African Journal of Botany 97 (2015) 1–8

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South African Journal of Botany

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Effects of storage under low temperature, room temperature and in the soil on viability and vigour of cordifolium () seeds

G.J. Brits a,⁎,N.A.C.Brownb, F.J. Calitz c, J. Van Staden d a Horticultural Division, Institute for Fruit Research, Agricultural Research Council, P.O. Box 5026, Stellenbosch 7599, South Africa b Conservation Biology Research Unit, National Botanical Institute, Kirstenbosch, P/Bag X7, Claremont 7735, South Africa c Biometry Services, Agricultural Research Council, P.O. Box 8783, Pretoria 0001, South Africa d Research Centre for Growth and Development, School of Biological and Conservation Sciences, University of Kwazulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa article info abstract

Article history: Seed longevity, the control of dormancy and the eventual fate of seeds were studied in two experiments simulat- Received 20 July 2014 ing conditions in natural fynbos. In one experiment a batch of mature, freshly harvested, intact achenes (“seeds”) Received in revised form 10 November 2014 of the myrmecochorous species Leucospermum cordifolium was divided into lots, of which one was buried in Accepted 12 November 2014 mesic mountain fynbos (experiencing a mediterranean-type climate with hot summers). Other seed lots were Available online xxxx stored open at room temperature and in closed nitrogen filled containers at 3 °C, respectively. Stored seeds Edited by MI Daws were sampled and germinated under optimal laboratory (viability estimate) and seed bed (vigour estimate in fynbos) conditions, during autumn, after 0 (control), 1, 2 and 4 years. Low temperature stored seeds maintained Keywords: a high viability and vigour for c. two years but ambient temperature storage led to a marked decline after 1 year Leucospermum ending in almost complete mortality after 4 years of shelf storage. Four year soil-stored seeds, by contrast, main- Dormancy tained a high viability and vigour, of 80% and 60% respectively of the original seed source values. The soft peri- Germination carp/elaiosome in soil-stored seeds disappeared completely whilst the testa became progressively scarified Persistent seed bank over time. The strongly increased germination rate (velocity) in soil-stored seeds was attributed to natural oxy- Soil-storage genation mediated by testa scarification. In another experiment freshly matured intact achenes were oxygenated Viability with 1% H O and the pericarps removed. Disinfection and benzyladenine growth regulator soaking (200 mg L−1 Vigour 2 2 Wet–dry cycling for 24 h) were applied as separate treatments. The seeds were then sown at 1 cm depth in a standard seed bed in autumn and germination was recorded in the first winter season and, in the undisturbed seed bed, in each sub-

sequent winter for 5 germination seasons. H2O2 oxygenated seeds gave a much higher germination percentage and rate than non-oxygenated seeds, although sporadic germination continued over five germination seasons in all treatments. Seeds germinated only during autumn and early winter each year. The results of the combined experiments suggest that, in nature, Leucospermum seeds can persist underground for long periods. We propose a model of soil-stored seeds in which the intact testa of freshly dispersed seeds is gradually scarified, leading to uneven, extended germination of the young (mainly current season) seed cohort over the first (and possibly several) post-fire germination seasons; and the synchronous germination of the older (scarified), larger, portion of the seed bank, leading to massive species recruitment during the early stages of the first post-fire winter germination season. The numerous wet–dry cycles due to natural rainfall, over prolonged periods in fynbos, may contribute to seed longevity via cellular repair processes. © 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction in Leucospermum R. Br. seed germination, all relating to dormancy control (Brits et al., 1993, 1999) make little sense without understanding longev- Dormancy and its control are mainly linked to longevity in seeds. ity of Leucospermum seeds during soil storage. Circumstantial evidence of Thus desiccation–scarification patterns, the stimulative effects of prolonged underground seed longevity in the myrmecochorous, nut- scarification/oxygenation and alternating temperature requirements fruited Proteaceae (to which Leucospermum belongs) is often seen in the literature (Rourke, 1972). However, systematic study of seed soil storage in Proteaceae and other taxa in fynbos and other types of medi- ⁎ Corresponding author at: Brits Nursery, 28 Flamingo Rd, Stellenbosch 7600, South terranean fire-prone vegetation is generally lacking. In fynbos, attrition Africa. Tel./fax: +27 21 8864710. E-mail addresses: [email protected] (G.J. Brits), [email protected] (N.A.C. Brown), of buried seed samples of 6 non-proteaceous species during soil storage [email protected] (F.J. Calitz), [email protected] (J. Van Staden). was rapid over 6–30 months, including those of myrmecochorous seeds

http://dx.doi.org/10.1016/j.sajb.2014.11.003 0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved. 2 G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8

(Pierce and Cowling, 1991). In California chaparral, soil-stored seed banks sowing in the natural autumn germination season (Brits and Van decay continuously, including those of fire-recruiting species (Parker and Niekerk, 1976). Kelly, 1989). Losses during soil storage are thought to be caused mainly It is believed that complex ecological–physiological interactions by pathogens (Cook, 1980). Germination during storage in unburnt occur in seeds under field, as opposed to laboratory, conditions vegetation (Cook, 1980; Pierce and Cowling, 1991) and seed ageing (Keeley et al., 1989). We simulated the burnt fynbos germinative envi- (Cavers, 1983) also lead to seedling loss. Soil-stored seed banks of four ronment of Leucospermum as closely as possible to augment laboratory chaparral shrub species were found not to accumulate steadily in the studies (Brits and Van Niekerk, 1976, 1986). To this end seeds were soil (Keeley, 1977). However, some species have seeds with a soil- germinated in an open seed bed situated within the fynbos habitat of storage life exceeding 20–100 years (Keeley, 1991). In the first artificial Leucospermum cuneiforme (Burm. f.) Rourke, an allopatric species of seed storage study in fynbos, Holmes and Newton (2004) showed that L. cordifolium, with a similar seed biology (e.g. growing on slopes of Leucospermum conocarpodendron (L.) H. Buek seeds have longterm shallow sandstone soil and at overlapping altitudes — Rourke, 1972) persistence and maintained a high viability, over three years of fynbos under late autumn conditions (Vogts, 1976). soil burial. Coupled to seed longevity in Leucospermum is the role of strategical- 2. Materials and methods ly extended germination (“germination polymorphism” sensu lato — Cavers and Harper, 1966; Keeley, 1991/“staged seed germination”— 2.1. Seed source Ayre et al., 2009) of seeds within the same (Brits, 1987) and over several germination seasons. These seed characters and their ecological Freshly matured achenes of L. cordifolium were produced at relationships are critical in short- and longterm avoidance of local dec- Tygerhoek Experimental Farm, Riviersonderend (34°9′S, 19°54′E) on imation of seedling populations in mediterranean-type ecosystems Table Mountain Sandstone derived soil within mesic mountain fynbos, (Bell et al., 1993; Keeley, 1991) and generally in moisture-stressed, un- and harvested under controlled conditions, after natural release in predictable environments (Gutterman, 1993). Extended germination December. These “seeds” were harvested from essentially non- has been poorly studied both in fynbos genera with soil-stored seeds domesticated as achenes, the true dispersal units, with a mature, (Holmes and Newton, 2004; Pierce and Cowling, 1991) and in soil- turgid pericarp (elaiosome) present (Brits et al., 1993) and were dried stored Proteaceae in Australian mediterranean-type vegetation (Ayre for 6 weeks under summer conditions in the shade. The dried seed et al., 2009; Lamont et al., 1985). However, observations of “erratic” batch was transferred to −10 °C for 48 h to disinfest seeds of possible (extended) germination of many Proteaceae species with nut-fruited granivores. Seeds were then hand sorted to ensure the use of fully seeds within the first germination season and even over subsequent developed embryos (Van Staden and Brown, 1973; Vogts, 1976) and seasons are well documented (e.g. Knight, 1809; Van Staden and thoroughly mixed. Brown, 1973). In the present study, storage under simulated fynbos seed bank 2.2. Seed storage experiment conditions was compared with the accepted commercial method of open-shelf storage at ambient temperature (R. Parsley, personal com- 2.2.1. Storage treatments munication) and a longevity increasing storage regime. Van Staden A batch of freshly matured intact seeds of L. cordifolium was divided (1978) stored seeds of neriifolia R. Br. (a fynbos Proteaceae into four lots and stored under four storage regimes during late species) for three years, with minimal loss of viability, at 5 °C and in summer: nitrogen gas at 20 °C. A combination of 3 °C in nitrogen was used in our study in an attempt to increase the storage life of Leucospermum 1. Control seeds were sown in a seed bed (see detailed methods in cordifolium (Salisb. ex Knight) Fourcade seed. Sections 2.2.2 and 2.2.3), following two months of dry storage at To test the survival capacity of stored seeds, viability and vigour ambient temperature and humidity (Year 0). were measured. Viability may be defined as the percentage seeds that 2. Soil-storage (simulating ant dispersal and pre-fire soil seed bank give rise to normal seedlings under optimal incubation conditions in a conditions): two hundred intact seeds were placed in a bag of glass standard laboratory germination test, and the biochemical tetrazoli- fibre gauze (1.5 mm mesh size), covered in addition by an outer um–formazan reduction colouring test may be used as an alternative layer of galvanized wire gauze (1 cm mesh size) to exclude possible viability test (International Seed Testing Association [ISTA], 1985). seed predators. Three such sub-lots were prepared and buried in fyn- Disinfected seeds of L. cordifolium which are laboratory incubated in bos soil. Seeds were buried in a typical L. cuneiforme habitat in mesic oxygen under favourable alternating temperatures (Brits, 1986a) fynbos (Brits, 1987), unburnt for eight years, in the zone where could lead to near-complete germination of the viable seed fraction, as L. cuneiforme seeds are normally dispersed by ants (Brits, 1987). A compared with the results of a tetrazolium colouring test (termed spot was chosen 75 cm south of each of three L. cuneiforme shrubs “laboratory” germination — Brits, 1990). The effect of dormancy in (reference plants) in average shade (Brits, 1986a) of the reference such seeds can thus be distinguished from the effects of other factors, and other plants such as Erica spp. A spade was inserted parallel such as non-viability (Brits et al., 2014). Another method of stimulating with the soil surface at c. 3 cm depth in the direction of the reference all viable, including dormant, seeds to germinate (to separate dormancy plant so as to minimally disturb the soil and root mass, the spade was effects) is the use of growth regulators in addition to an optimizing en- lifted slightly and the fl at bag with L. cordifolium seeds guided into vironment. Cytokinin, for example, was found to enhance Proteaceae place after which the soil mass (damp from recent rain) was replaced seed germination when applied exogenously at a low concentration as before. (Brown and Van Staden, 1973) and in this study cytokinin was applied Soil-storage treatments were limited to a single seed sub-lot as the commercial preparation benzyladenine. (replicate) per uninterrupted storage period on account of a scarcity Seed vigour may be defined as those qualities which determine of reference plants and the need to leave soil-stored seeds in an rapid, uniform emergence of normal seedlings (of domesticated crops) undisturbed condition in each storage site, which precluded the under a wide range of field conditions (McDonald, 1980). The vigour option of periodic sub-sampling from each bag. of dormant seeds ageing and declining in the fynbos soil-stored seed The seed bag sub-lot from each reference plant was removed after bank would be an important parameter in the study of natural seedling one, two or four years of undisturbed burial and the seeds examined recruitment. In order to test the vigour of the Leucospermum seed source under a binocular microscope for signs of possible damage and mor- whilst removing the potentially confounding effect of seed coat anoxia tality. Empty, broken testas were assumed to indicate germinated dormancy, seeds may be oxygenated with 1% H2O2 before seed bed seeds and were counted. The seed coat was inspected for signs of G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8 3

scarification brought about by soil factors such as microbial decom- bed emergence were calculated by means of the formula of Heydecker position. Unbroken seeds were removed and cleaned slightly, taking (1973) (Brits et al., 2014): care not to add to the degree of scarification already present. 0 1 Xk 3. Warm-storage (dry storage under ambient conditions): Seeds were B C B ni C stored in groups of 33 in porous plastic bags placed in a 1 L glass B ¼ C fl fi GR ¼ B i 1 C:100 ask of which the opening was covered with a single layer of lter B Xk C fl @ : A paper. The ask was placed on a laboratory shelf in shade and at Di ni annual intervals seed bags were removed randomly from the flask i¼1 for germination tests. 4. Cold-storage (dry storage at 3 °C in nitrogen): Seeds were stored in where groups as above in a 1 L glass flask in nitrogen and closed airtight with a screw-on lid sealed with petroleum jelly. The flask was k final week of germination flushed at six-monthly intervals. At designated intervals seed bags Di number of weeks from sowing were taken randomly from the flask for germination tests after ni number of seeds newly germinated in week Di standing the flask at room temperature to attain temperature iweek1toweekk. equilibrium. The experimental design was a completely randomized design 2.2.2. Storage periods, seed preparation and sowing with 24 treatments with 3 random replications. An experimental unit Seeds were germinated in March–April in Years 0, 1, 2 and 4. Har- consisted of 20–33 seeds. The treatment design was a 2 × 3 × 4 factorial vested intact achenes were soaked in distilled water and in non-soil with factors: 2 regimes (laboratory germination and seed bed emer- stored (non-buried) seeds the soft pericarp was then removed (Brits gence); 3 treatments (soil-, warm-, cold-stored); and 4 storage periods et al., 1993). The resulting seeds proper were dried lightly with paper (Year0=start;1,2,4yearslater). towels and treated with thiram wettable powder fungicide (Benic, The data were subjected to an appropriate factorial analysis of 1986). In scarified soil-stored seeds care was taken not to disturb the variance (ANOVA). The Shapiro–Wilk's test was pre-performed on the generally fragile condition of the covering structures (see Section 3.1.3). standardized residuals to test for deviations from normality (Shapiro and Wilk, 1965) and as there was not sufficient evidence against nor- 2.2.3. Germination regimes mality, the data were considered statistically reliable (not needing Experimental seeds were germinated under two regimes to com- transformation). Student's t-LSD (least significant difference) was cal- pare their viability and vigour, respectively. culated at a 5% significance level to compare means of significant source effects. All the above data analyses were performed with SAS version 6 1. Laboratory germination to test viability. Seeds were dark-incubated statistical software (SAS, 1988). in medical grade (N99% pure) oxygen in 1 L flasks, on a single layer of Whatman No. 1 filter paper (Brits, 1990) under a favourable 2.2.5. Microscopy alternating 8 °C for 16 h × 24 °C for 8 h, daily, temperature regime Soil-stored seeds for SEM examination were freeze-fractured in for L. cordifolium (Brits, 1986a); flasks were flushed with oxygen liquid nitrogen, mounted onto aluminium stubs and sputter coated twice per week. with gold–palladium for viewing in a Cambridge S200 at 10 kV. A tetrazolium viability test was carried out in addition, in Year 0, on a sample of seeds, to obtain an alternative estimate of the viability of 2.3. Extended germination of L. cordifolium seeds in the seed bed the fresh seed source (Brits and van Niekerk, 1976). Six replicates of 33 seeds were treated and scored and the mean tetrazolium Fresh, mature, dry, intact seeds of L. cordifolium, using the same seed colouring values calculated. source as above, were disinfected in hot water at 50 °C for 30 min 2. Seed bed germination (i.e. seedling emergence) to test vigour. Seed (Benic, 1986). Seeds were soaked in 1% H2O2 for 24 h (Brits, 1986a)or samples from storage treatments were not oxygenated and were − in a 200 mg L 1 benzyladenine (BA) solution (Mitchell et al., 1986)or germinated at 1 cm depth in standard acidic sand seed beds, located in a combination of BA and H O . Controls were soaked in water, one within a mesic fynbos environment (see locality detail in Section 2.1) 2 2 disinfected, not receiving H O oxygenation or BA treatment, and another and fully exposed to sunlight, annually in autumn (Vogts, 1976 — i.e. 2 2 non-disinfected and similarly treated. The softened pericarps were re- at the start of the mediterranean-type rainy season). Seed beds were moved (Brits et al., 1993) and the resulting seeds proper were washed disinfected because regular use can cause accumulation of pre- and briefly in running tap water and then allowed to dry until the seed coat post-emergence seed(ling) pathogens (Benic, 1986). Disinfection contained no free water. Seeds were then treated lightly with thiram fun- consisted of spray-watering the sand medium in the bed with a 3% − gicide powder and sown in autumn as in the storage experiment (see formalin solution at an application rate of 10 L solution·m 2. Seed Section 2.2.3). Six replicates of 33 seeds per treatment were sown in a beds were irrigated daily from the time of sowing. randomized block design and seeds were left in situ, undisturbed, for To estimate germination vigour in the fresh seed source (non- five years. Seed bed emergence was recorded weekly throughout the stored), the pooled seed bed emergence scores of the two H2O2 oxygen- winter (20 weeks) in the first and over four successive winters; observa- ated treatments in the extended-germination experiment in Section 2.3 tionsofweeks8–20 were pooled at the end of each season. Seed bed ir- were used. The same seed source was used and the two experiments rigation was discontinued during the summer months and the seed bed commenced on the same date. was weeded at the start of each autumn germination season to obtain a sun-exposed soil surface. ANOVA was performed on final germination 2.2.4. Observations and statistical treatment (emergence) percentages and rates as in the storage experiment. Germination and emergence in Experiment 1 were recorded weekly for 12 weeks, until germination stabilized in mid-winter, thus providing 3. Results both cumulative and final germination percentage responses. Laborato- ry incubated seeds were scored as germinated when the radicle had 3.1. Seed storage experiment protruded at least 1 mm. In the seed bed successful emergence was re- corded when the seedling cotyledons had unfolded aboveground. The The tetrazolium viability estimate for the seed source at the start of rates (GR — coefficient of velocity) of laboratory germination and seed the study was 81% (Fig. 1a). In contrast H2O2 oxygenated seeds in the 4 G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8

Seeds cold-stored in nitrogen, germinated to the level of maximum viability over Years 1 and 2, close to the level of the tetrazolium viability estimate and the initial value of unstored seeds in Year 0 (Fig. 1a). In contrast seed bed germination (emergence) of cold-stored seeds rose markedly from Year 0 (fresh seeds) to Years 1 and 2, the Year 2 differ- ence being statistically significant (P b 0.05 — Fig. 1b). The decline of cold-stored seed bed emergence in Year 4 corresponded with a decrease in laboratory germination, both changes from Year 2 being statistically significant (P b 0.05). In soil-stored seeds both germination and emergence were poor in Years 1 and 2, then increased to near original levels in Year 4, the latter differences in the case of seed bed emergence values not being statisti- cally significant (Fig. 1). Soil germinated seeds (broken seed coats) amounted to 6% (Year 1), 6% (Year 2) and 12% (Year 4). Under both regimes the Year 4 buried seed values exceeded the performance of cold-stored seeds, although the differences were not statistically significant (Fig. 1). The Year 4 soil-storage values represent c. 80% of the original tetrazolium and germination viability estimates (Fig. 1a) and c. 60% of the emergence vigour estimate (Fig. 1b). Storage at ambient temperature and humidity (warm-storage) led to a much faster decrease in germination/emergence percentages than low temperature (3 °C) storage (Fig. 1). Thus in Year 1 warm-stored seeds germinated and emerged on a par with Year 0 and cold-stored seeds but surprisingly soon, in Year 2, germination/emergence fell markedly (P b 0.05 — Fig. 1). Seed germination/emergence occurred only at extremely low levels in both the laboratory and seed bed regimes after four years.

3.1.2. Rates (GR — velocity coefficient) of germination and emergence

Fig. 1. Laboratory germination in oxygen (a) and seed bed emergence (non-oxygenated) Laboratory regime germination rates were markedly higher than (b) percentages of L. cordifolium seeds stored in the soil (· · ·◊· · ·), under ambient seed bed regime emergence rates (Fig. 2,Pb 0.001 — Table 1). In the temperature (- - ○ - -) and at 3 °C in nitrogen (—Δ—) for various periods; - - - tetrazolium seed bed seedlings were observed to emerge at least 1–2 w slower viability estimate (a) or seed bed vigour estimate (of H2O2 oxygenated seeds) (b). Bar than seeds germinating in the laboratory. represents LSD (P = 0.05). Buried seeds germinated much faster than non-buried seeds especially after two and four years of soil-storage (Fig. 2). Velocity in- seed bed (in the extended-germination trial) averaged 57% emergence creased over time, illustrated by the positive slopes of both laboratory in Year 0 plus a further 13% emerged over the following 4 years, totalling germination (b = 13.261; P b 0.05 — Fig. 3) and seed bed emergence

70% (see H2O2 oxygenated values in Fig. 5). The latter figure was used as (b = 10.579; P b 0.01 — Fig. 3) in the regression of rate on period of an estimate of the vigour of the seed source (Fig. 1b). soil-storage. The GR in non-buried seeds, sown with intact testas, did notdiffersignificantly within either the seed bed or the laboratory, except in cold-stored seeds of Year 2 in the laboratory regime (Fig. 2a). 3.1.1. Germination and emergence percentages Seed bed regime emergence was consistently lower than labora- 3.1.3. Microscopy tory regime germination, the average difference totalling 33% (Fig. 1; Effective scarification was confirmed by inspection (Fig. 4) revealing Table 1) with the two regimes showing similar treatment patterns, no remnants of the pericarp/elaiosome on buried seeds and with the i.e. with low statistical interaction (Regime × treatment — Table 1). exotesta extensively scarified by Year 4 (Fig. 4a). The woody endotesta surprisingly also became brittle and easily fi Table 1 breakable by nger pressure, which was caused by cracking (Fig. 4b) in- Analysis of variance of L. cordifolium seed germination rates and percentages under dicating further strong scarification. Mechanically and visually the testa different storage treatments, periods (years of storage) and germination regimes. after 2 and 4 years of soil-storage more or less resembled acid scarified seeds (Brits and Van Niekerk, 1976). Significantly the embryos in soil- Source d.f. Germination rate Germination (GR) percentage stored seeds up to Year 4 appeared white, plump and healthy.

MS SL MS SL 3.2. Extended germination experiment Regime 1 3527.623 0.0001 17520.335 0.0001 Treatment 2 3615.490 0.0001 2848.021 0.0001 Reg ∗ Treat 2 401.811 0.0239 22.582 0.6952 Exogenous benzyladenine did not affect seedling emergence Period 3 2500.759 0.0001 1260.876 0.0001 percentage (Fig. 5), therefore the +BA and −BA treatments are treated ∗ Period Reg 3 235.010 0.0823 499.801 0.0002 as two replicates of the other factors, H2O2 oxygenation and disinfec- Period ∗ Treat 6 1291.732 0.0001 2878.270 0.0001 tion, this being consistent with Brown et al. (1986) and Brits et al. ∗ ∗ Period Reg Treat 6 284.100 0.0190 174.777 0.0207 (1995) who found BA not to stimulate laboratory germinated Error 42 98.369 – 61.582 – Total 65 ––––L. cordifolium seeds. Disinfected seeds showed a marked lead over the control (P b 0.05) in the first germination season and maintained Abbreviations this up to the 5th germination season (Fig. 5). MS Mean square d.f. Degrees of freedom H2O2 oxygenated seeds gave a much stronger response, in both SL Significance level cumulative emergence percentage and emergence rate, than control G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8 5

Fig. 4. Effective scarification of the testa in L. cordifolium seeds after 4 years of soil-storage; the exotesta (a) with its overlying pericarp completely decomposed, is ruptured extensively, exposing the endotesta. Hairline cracks in the endotesta (b) contribute to a brittle seed coat texture. en — endotesta (palisade cells); ex — exotesta. Fig. 2. Rates (GR — coefficient of velocity) of laboratory germination (a) and seed bed emergence (b) of L. cordifolium seeds stored in the soil (stippled bars), under ambient temperature (hatched bars) and at 3 °C in nitrogen (solid bars) for various periods; small bar represents LSD (P = 0.05). Missing bars are due to non-calculable germination/ season (cf. Brits and Van Niekerk, 1976). Control seeds gave the typically emergence values. erratic, extended pattern, and low total emergence, of intact non- oxygenated seeds (Brits and Van Niekerk, 1976), thus totalling only seeds (Fig. 5). Seedling emergence percentages of oxygenated seeds ac- 34% emergence in the first year of the final portion emerged (by the cumulated close to the tetrazolium viability estimate by the 5th year, end of the 5th season), amounting to only 16% of the vigorous seed suggesting near-complete emergence of vigorous seeds after the 5th

Fig. 5. Seed bed cumulative emergence percentages of L. cordifolium seeds extending over

5 germination seasons; —□— H2O2 oxygenated; — — H2O2 + benzyl adenine (BA) Fig. 3. Regressions of laboratory germination rate (●, —) and seed bed emergence rate treated; —▲— disinfected + BA; —■— disinfected; —◊— control; - - - tetrazolium viability (○,---)onperiodofsoil-storageinL. cordifolium. Germination rate: Y = 35.104X + estimate (ex storage experiment); bar represents LSD (P = 0.05) for final percentages. 13.261; R2 = 0.51 (laboratory). Emergence rate: Y = 27.274X + 10.579; R2 =0.79 Emergence was recorded weekly; the last data points in each germination season (seed bed). represent the total emergence for weeks 8–20. 6 G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8

fraction (i.e. of the cumulative total of H2O2 oxygenated seeds emerged the endotesta would have permitted rapid emergence of the radicle, by the end of the 5th season — Fig. 5). H2O2 oxygenated seeds, by which may have contributed to the high GR of buried seeds. Alternative- contrast, gave 83% emergence (average of two H2O2 treatments) in ly seeds may have completed and accumulated the initial stages of ger- the first germination season, increasing to 100% of vigorous seeds (by mination during repeated wet periods in nature, shortening the final definition) by the fifth season. Emergence in all treatments extended period of continuously favourable conditions required for germination over five germination seasons. (Baker et al., 2005; Lush et al., 1981).

4. Discussion 4.3. Seed bed vs. soil-storage conditions

4.1. Seed viability and vigour The rapid germination of soil-stored seeds following sowing in the seed bed indicates that seed dormancy effectively enforced over 4 The constantly lower performances in both final germination years in the reference plant soil zone, must have been removed under percentage (Fig. 1)andGR(Fig. 2) of seeds in the seed bed as opposed seed bed conditions (Fig. 2). Factors that may have contributed to this to the laboratory reflect the difference between seed vigour and seed are 1) removal from allelopathic suppression of germination by the ref- viability measurement. Viability estimates over Years 0 and 1 for non- erence (and other) plants (enforced dormancy — Keeley and Keeley, buried seeds corresponded closely with the tetrazolium viability 1989); 2) removal from oxygen deprivation by respiring roots and soil estimate (Fig. 1a) validating the use of the laboratory oxygenation ger- microflora (Brits, 1986b); and 3) most likely, the strongly increased di- mination regime as a measure of viability. In the seed bed, on the other urnal temperature amplitude in the exposed seed bed surface soil, as hand, considerable embryo and seedling vigour, and more time, were opposed to the unburnt habitat (Brits, 1986a, 1987). In four year buried required in addition to radicle protrusion to a) establish an initial root seeds, the close-to-maximal germination and viability scores (Fig. 1a) system, b) displace a considerable weight of soil above the seedling suggest no need for further dormancy breaking mechanisms, e.g. and c) to expand the cotyledons at the soil surface. Marked differences smoke. Similarly, smoke did not promote germination of artificially may occur between seed viability and vigour estimates, especially in buried L. conocarpodendron seeds (Holmes and Newton, 2004), and seed populations with poor viability (Hegarty, 1974; Moore, 1973). was not found to be a ubiquitous requirement for dormancy breaking Seed bed emergence also declined faster than laboratory germina- in Leucospermum (Brown and Botha, 2004). Secondary dormancy in tion, as is reflected by Year 2 values of warm-stored seeds (Fig. 1), illus- the immediate post-burial/post-sowing phase is not ruled out (Figs. 1 trating that vigour of a seed population declines faster than its viability and 5), although our scarification–oxygenation–alternating temper- (Delouche and Caldwell, 1960). Seed bed emergence (vigour depen- ature model seems to sufficiently explain dormancy breaking in dent) of non-oxygenated seeds, both cold and warm-stored, would in Leucospermum (Brits, 1990; Brits et al., 2014). addition have been influenced by testa imposed dormancy effects. In cold-stored seeds the increase in seed bed emergence over Years 4.4. Decline in germinability of non-buried seeds 0–2(Fig. 1b) would not have been due to after-ripening (Brits and Van Niekerk, 1986) and may reflect variations in yearly seasonal conditions Non-buried seeds declined rapidly (for seeds suspected to be long- expressed in the seed bed, but not in the laboratory (see seed bed lived), and this was marked even at low temperature in a nitrogen at- germination, Section 2.2.3). mosphere. Bass (1980) concluded that seed storage under inert gases is not effective and that the main determinants of longevity are humid- 4.2. Responses of soil-stored seeds ity and temperature. Considerable early work on seed ageing in fynbos Proteaceae by Van Staden and his co-workers includes Van Staden A limitation of our soil-storage study was that neither natural varia- (1978, 1981) who reported deterioration patterns of P. neriifolia seeds tion nor time trends could be determined, due to a lack of experimental during longterm dry storage. Even when stored at −10 °C seeds replication, however the burial treatments represent single-sample showed signs of deterioration and seeds stored at 20 and 26 °C showed estimates of year effects on germinability of soil-stored seeds. Thus primarily lipid body and lipid membrane degeneration (Van Staden, the significantly higher germination percentage of buried seeds in 1978, 1981), but also deterioration of protein bodies and nuclear mate- Year 4 than in other storage years, although chance dependent, repre- rial. Damage to the latter in R. Br. affects glyoxysome sents a single unbiased estimate of burial effects in fynbos after 4 production and thus lipid utilization for energy release (Van Staden years (Fig. 1). In Years 1–2 seeds germinated/emerged in concert, et al., 1976) which is important as lipids are a primary storage reserve anomalously low relative to Year 4 values, and at variance with the in a number of Proteaceae seeds including L. cordifolium (Mitchell, conclusion of Cavers (1983) that viability declines continuously in the 1983; Van Staden et al., 1975, 1976, 1981). These reports are consistent soil-stored seed bank (Fig. 1). The low numbers of broken seed coats with research in other taxa with lipid-rich seeds (Jyoti and Malik, 2013; (i.e. soil germinated seeds — Section 3.1.1)ruleout“pre-fire” germina- Shaban, 2013). In soybeans, which also contain a relatively large tion, due to a lack of dormancy, as a cause of the anomalies. Low germi- amount of storage lipid, the loss of both seed viability and vigour was nation is possibly due to unknown experimental artefacts, e.g. localised slowed when seeds were hydrated, then dehydrated, during mid-term fungal/microbial infection during soil storage — these could not be storage (Saha et al., 1990) and the proposed mechanism for this phe- detected in our single replication experiment. However the high Year nomenon is the activation of the cellular repair system and counterac- 4 estimate is probably the best indicator of potential seed longevity dur- tion of free radical and lipid peroxidation reactions during hydration. ing natural soil storage, assuming that adaptedness to myrmecochory in Long et al. (2011) showed that the repeated wet–dry cycles of natural fynbos would be more effective in regulating seed survival than our rainfall extended longevity in field-buried Avena sterilis seeds via re- artificial treatments. instatement of antioxidant systems. The strong increase in GR (velocity) over time (soil-storage period — A similar model applies to the degradation of DNA where seeds Fig. 3) suggests time dependent scarification of the testa during soil- employ repair mechanisms in the early stages of seed germination storage, equating with a regulatory role for improved oxygenation. (Boubriak et al., 1997; Rajjou et al., 2012; Waterworth et al., 2010), Increased GR is consistent with the advanced scarification evident in but also in imbibed dormant seeds (Baskin and Baskin, 2014). Conse- four year buried seeds (Fig. 4). In freshly matured, intact seeds the quently, the numerous hydration/dehydration cycles, and therefore exotesta is the main component imposing anoxia dormancy (Brits opportunities for cellular repair, to which dormant L. cordifolium soil- et al., 1999) and the exotesta extends uniformly and unbroken over stored seeds were subjected to in nature, may explain the differences the entire seed surface (Manning and Brits, 1993). The brittle state of in response between soil- and warm-stored seeds. G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8 7

4.5. Extended germination 2) the seed coat layers are progressively scarified during soil storage and conditions in unburnt fynbos, typically at 3 cm burial depth, In both our trials, seed bed emergence probably reflects population enforce dormancy indefinitely on seeds; recruitment patterns in nature. Thus, in fynbos some young (freshly 3) only co-occurring oxygenation and alternating temperatures are dispersed, current-season), intact seeds would, after fire, show the ex- subsequently required to break dormancy; tended emergence pattern of non- or weakly scarified control seeds in 4) in nature the gradual progress of scarification of freshly matured the extended-germination trial whereas older, strongly scarified soil- seeds, implies a low GR of young seeds, i.e. that after fire primarily stored seeds would show the rapid emergence pattern of oxygenation the young-seed cohort will germinate unevenly over an extended treatments. This model parallels the genetic inference of Ayre et al. period of time — whereas the larger, older, i.e. well-scarified, portion (2009) in a mollis (Proteaceae) population that a second of the seed bank will germinate rapidly and synchronously in a recruiting seedling cohort comprised younger seeds, after chance fire major, first, wave of recruitment; destroyed the first recruiting cohort, the latter being associated with 5) the water-permeable seeds can, in nature, tolerate repeated hydra- the older portion of the soil stored seed bank. tion and desiccation whilst maintaining high viability; regular A pattern of seedling cohorts emerging in succession over one or wet–dry cycles may indeed be the key to maintaining viability. We several germination seasons (Fig. 5) could result in the extant gene speculate that this feature of soil storage could involve activation pool escaping local extinction through intermittent disturbance, e.g. of cellular repair mechanisms during wet, dormant periods. by predation (Cavers, 1983) and short inter-fire periods (Ayre et al., 2009). Extended emergence patterns were observed in post-fire Acknowledgement recruiting L. cordifolium and L. cuneiforme seedling populations in fynbos (Brits, 1987), this being limited to a minority of seedlings, in contrast to The financial support for this research by the National Vegetable and a large majority of even-aged seedlings which emerged early in the Ornamental Plant Institute (ARC) under Project GS2431/30/2/4, is season (Brits, 1987). gratefully acknowledged and particularly the technical assistance of Disinfected seeds emerged much better than control seeds (Fig. 5) Mr. G. van den Berg of the Institute. and it is not known whether suppression of pathogens, or possibly an unknown treatment artefact caused the difference (however direct heat-mediated physiological stimulation of the embryo during disinfec- References tion may be excluded — Brits et al., 1999). Resumption of germination Ayre, D.J., Ottewell, K.M., Krauss, S.L., Whelan, R.J., 2009. Genetic structure of seedling only during autumn in successive years (Fig. 5) is consistent with the cohorts following repeated wildfires in the fire-sensitive shrub Persoonia mollis ssp. – narrow alternating germination temperature requirements proposed nectens. Journal of Ecology 97, 752 760. Baker, K.S., Steadman, K.J., Plummer, J.A., Merritt, D.J., Dixon, K.W., 2005. The changing for Leucospermum (Brits, 1986a; Brits et al., 2014). window of conditions that promotes germination of two fire ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae). Annals of 4.6. 200 year-old germinable Leucospermum seed? Botany 96, 1225–1236. Baskin, C.C., Baskin, J.M., 2014. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. 2nd Edition. Academic Press (Elsevier), San Diego (Ch. 7). Daws et al. (2007) found a single Leucospermum seed out of 61 Bass, L.N., 1980. Seed viability during longterm storage. Horticultural Reviews 2, 117–141. Proteaceae seeds (14 of which were Leucospermum seeds) that germi- Bell, D.T., Plummer, J.A., Taylor, S.K., 1993. Seed germination ecology in southwestern – ≥ Western Australia. Botanical Review 59, 24 54. nated at 20/10 °C alternating temperature after shelf storage for 203 Benic, L.M., 1986. Pathological problems associated with propagation material in protea years at an estimated average of 15–18 °C and 50% RH. They suggest nurseries in South Africa. Acta Horticulturae 185, 229–236. that possible long-term natural seed survival in Leucospermum may be Boubriak, I., Kargiolaki, H., Lyne, L., Osborne, D.J., 1997. The requirement for DNA repair in – linked to an oxygen-impermeable testa and that their result may indicate desiccation tolerance of germinating embryos. Seed Science Research 7, 97 105. Brits, G.J., 1986a. Influence of fluctuating temperatures and H2O2 treatment on germina- adaptation for extreme longevity in Leucospermum. The drastically differ- tion of Leucospermum cordifolium and florida (Proteaceae) seeds. South ent outcome of the present study shows complete deterioration of seeds African Journal of Botany 52, 286–290. after 4 years on the shelf, and in an oxygen-free environment, at constant Brits, G.J., 1986b. The effect of hydrogen peroxide treatment on germination in Proteaceae species with serotinous and nut-like achenes. South African Journal of Botany 52, 3 °C, there was also marked deterioration after 4 years. Importantly our 291–293. current model requires, in nature, wet–dry cycles (regular wetting most- Brits, G.J., 1987. Germination depth vs. temperature requirements in naturally dispersed seeds of Leucospermum cordifolium and L. cuneiforme (Proteaceae). South African ly during the cool rainy season) for cellular repair to take place and thus – — Journal of Botany 53, 119 124. to maintain longevity in seeds (Boubriak et al., 1997; Long et al., 2011) Brits, G.J., 1990. Techniques for maximal seed germination of six commercial this was absent under all of the above shelf storage conditions. Leucospermum R. Br. species. Acta Horticulturae 264, 53–60. In Leucospermum embryo anoxia imposed by the intact exotesta has Brits, G.J., Van Niekerk, M.N., 1976. Opheffing van saadrus by Leucospermum cordifolium (Proteaceae). Agroplantae 8, 91–94. been shown only in the hydrated state (Brits et al., 1999; Van Staden Brits, G.J., Van Niekerk, M.N., 1986. Effects of air temperature, oxygenating treatments and and Brown, 1973) and this precludes an untested deduction about oxy- low storage temperature on seasonal germination response of Leucospermum gen diffusion in dried–stored seeds. Moreover in nature the oxygen ex- cordifolium (Proteaceae) seeds. South African Journal of Botany 52, 207–211. Brits, G.J., Calitz, F.J., Brown, N.A.C., Manning, J.C., 1993. Desiccation as the active principle cluding intact exotesta has a relatively short lifespan (this study; Brits in heat-stimulated seed germination of Leucospermum R. Br. (Proteaceae) in fynbos. et al., 1993, 1999) exposing the embryo to extraneous oxygen diffusion New Phytologist 125, 397–403. at an early stage. Our study shows that shelf-storage is an artificial con- Brits, G.J., Cutting, J.G.M., Brown, N.A.C., Van Staden, J., 1995. Environmental and hormonal regulation of seed dormancy and germination in Cape fynbos Leucospermum R. Br. dition, without a natural parallel, that quickly destroys longevity. In fyn- (Proteaceae) species. Plant Growth Regulation 17, 181–193. bos the question of extreme longevity of soil-stored seeds therefore Brits, G.J., Calitz, F.J., Brown, N.A.C., 1999. Heat desiccation as a seed scarifying agent in goes unanswered, and caution should best be exercised in extrapolating Leucospermum (Proteaceae) and its effects on the testa, viability and germination. – the results of Daws et al. (2007) to fynbos. We think that (even short- Seed Science and Technology 27, 163 176. Brits, G.J., Brown, N.A.C., Calitz, F.J., 2014. Alternating temperature requirements in term) follow-up work would be desirable. Leucospermum R. Br. seed germination and ecological correlates in fynbos. South African Journal of Botany 92, 112–119. 5. Conclusions Brown, N.A.C., Botha, P.A., 2004. Smoke seed germination studies and a guide to seed propagation of plants from the major families of the Cape Floristic Region, South Africa. South African Journal of Botany 70, 559–581. Our results show that in Leucospermum,infynbos: Brown, N.A.C., Van Staden, J., 1973. The effect of scarification, leaching, light, stratification, oxygen and applied hormones on germination of Protea compacta R. Br. and daphnoides Meisn. South African Journal of Botany 39, 185–195. 1) undisturbed seeds in the soil are long-lived and may retain high viabil- Brown, N.A.C., Van Staden, J., Jacobs, G., 1986. Germination of achenes of Leucospermum ity and vigour for at least four years (at least at our experimental site); cordifolium. Acta Horticulturae 185, 53–59. 8 G.J. Brits et al. / South African Journal of Botany 97 (2015) 1–8

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