Influence of fluctuating temperatures and H202 treatment on germination of cordifolium and florida () seeds

G.J. Brits Vegetable and Ornamental Research Institute, Pretoria

Seeds of Leucospermum cordifolium (Salisb. ex Knight) Introduction Fourcade and Knight were germinated at 'Seeds' (achenes) of Leucospermum cordifolium (Salisb. ex constant temperatures of 4, 11, 18 and 25°C and also at Knight) Fourcade are dispersed annually at the end of the fluctuating temperatures that were made up of pairs of these temperatures over a 24 h period, the lower flowering season in mid-summer. They are released for many temperature of each pair being maintained for 16 h. An years after plants reach the flowering stage in their third or oxygenating treatment of soaking seeds in 1% H20 2 fourth year (Rourke 1972), apparently resulting in the accumu­ resulted in a slight increase in germination of L. cordifolium lation of a seed reserve in the soil. Seeds will apparently not seeds, without statistical interaction with temperature germinate in undisturbed vegetation until it is destroyed by conditions. Diurnal temperature amplitude had the strongest fire, after which dense, even-aged populations of seedlings effect on germination. An optimum low and high temperature of 9 and 24 oc respectively was calculated for emerge. This phenomenon has remained largely unexplained germinating seeds of L. cordifo/ium and 7 and 20°C for S. for L. cordijolium and other members of the Proteaceae with florida seeds. The daily surface soil temperature hard, nut-like seeds. The direct heat from fire apparently does fluctuations recorded in burnt mesic mountain not break dormancy in these seeds (unpublished results) unlike during winter was found to be of the same order as that in many members of the Leguminosae (Villiers 1972). required for germination. The temperature conditions Since seeds are insensitive to light (Horn 1962; Brown & Van recorded in unburnt, lightly or heavily shaded soils did not meet the germination requirements. A close relationship is Staden 1973), the improved illumination of burnt exposed soil therefore indicated between ecological conditions and would not influence germination. germination requirements in these two species. The breaking of dormancy in L. cordijolium seeds was S. Afr. J. Bot. 1986, 52: 286-290 shown to be strongly correlated with low seasonal air tempera­ ture (Brits & Van Niekerk 1986). Low temperature require­ Sade van Leucospermum cordifo/ium (Salisbury ex Knight) ment alone, however, does not explain the recruitment of Fourcade en Serruria florida Knight is by konstante temperature van 4, 11, 18 en 25°C en by wisselende seedlings en masse only after the occurrence of fire . temperature, opgemaak uit pare van hierdie temperature oor The seeds of some species are known to germinate 'n 24 h periode, ge"lnkubeer. By wisselende temperature is only under widely fluctuating diurnal temperatures (Koller die laer temperatuur vir 16 h gehandhaaf. Oksiginerende 1972). Proposed ecological functions for this requirement behandeling deur sade in 1% H20 2 te week het tot 'n include a sensor system to ensure that waterside plants would geringe toename in kieming by L. cordifolium sade gelei, germinate only when stranded after a period of floating sonder statistiese wisselwerking met temperatuur-toestande. Daaglikse temperatuurskommeling het kieming die sterkste (Thompson 1974) and for recognizing newly formed light gaps be"lnvloed. 'n Optimum lae en hoe temperatuur van 9 en in the forest canopy (Vazques-Yanes & Orozco-Segovia 1982). 24 oc respektiewelik is vir kiemende sade van L. cordifolium Temperatures of 10°C for 16 h alternated by 20°C for 8 h bereken en 7 en 20°C vir sade van S. florida. Die daaglikse were found to be a more suitable regime for germinating the wisseling in oppervlakgrondtemperature, gemeet in seeds of Proteaceae than either constant temperatures or gebrande mesiese bergfynbos, was gedurende die winter higher alternating temperatures (Brown & Van Staden 1971, van dieselfde orde as die benodig vir kieming. Temperatuur­ toestande gemeet in nie-gebrande, lig- of swaar-beskadude 1973). The purpose of this study was to determine whether grond het nie aan die temperatuurvereistes voldoen nie. 'n seeds of L. cordijolium and Serruria florida Knight required Noue verband bestaan dus tussen ekologiese toestande en high temperature in addition to the known requirement for kiemingvereistes by hierdie twee spesies. low temperature in order to break dormancy. An answer to S.-Afr. Tydskr. Plantk. 1986, 52: 286-290 the problem would also have commercial implications since these two species are widely cultivated from seed for the Keywords: Diurnal thermoperiodicity, dormancy, ecology, production of cut flowers (Vogts 1976). germination, Proteaceae Hydrogen peroxide may have an oxygenating effect when Submitted in partial fulfilment of an M.Sc. degree at the imbibed by achenes of Proteaceae species (Brown & Dix 1985). University of Stellenbosch (Botany Department) It was found to have an effect on germination in L. cordi­ folium similar to that of other oxygenating treatments, e.g. G.J. Brits Vegetable and Ornamental Research Institute, Private Bag X293, scarification (Brits & Van Niekerk 1976). Increasing the Pretoria, 0001 Republic of oxygen tension with H202 in terminating L. cordifolium seeds strongly influenced germination, independently from seasonal Accepted 14 February 1986 air temperature (Brits & Van Niekerk 1986). The interaction S. Afr. J . Bot., 1986, 52(4) 287

between various temperature regimes and increased oxygen sunshine in midsummer was measured at: tension was also investigated in the present study. burnt site: 128,69 103 lux lightly shaded site: 28,12 103 ± 11 ,71 103 lux Materials and Methods heavily shaded site: 5,72 103 ± 2,07 103 lux Germination experiment Thermometers were placed at a depth of 5 mm in the soil Seeds harvested freshly from cultivated L. cordifo!ium and of the burnt exposed fynbos or in litter under plants. S. florida plants were treated with hot water (55°C) for 20 Maximum and minimum soil temperatures were recorded min to reduce microbial growth on seeds (Bertie & Knox­ during five days per week in each of the sites from April1983 Davies 1983). Seeds were soaked in either distilled water or to July 1984. a 1OJo H20 2 solution for 24 h at 22°C before incubation (Brits & Van Niekerk 1976). After soaking, the gelatinous outer Results and Discussion pericarp of L. cordifo!ium seeds was rubbed off and seeds Analysis of transformed [angular transformation to correct were washed for 30 s in running tap water before incubation. non-normality in proportions (Snedcor & Cochran 1967)] Seeds were germinated at ten different temperature regimes. percentage data showed that, for seeds of L. cordifolium, Where temperatures were alternated the lower and higher treatment with H202 led to significantly (P < 0,05) improved temperature was maintained for 16 and 8 h respectively. The germination over untreated seeds. Mean germination in treated regimes were: and untreated seeds was 28,6 and 22,8% respectively (compare 1. 4°C constant 6. l1 °C x 18°C Figure 1A and 1B). The statistical interaction of H202 x 2. 11 oc constant 7. 18°C x 25 °C temperature treatments was not significant (P > 0,25). For 3. 18°C constant 8. 4°C x 18°C seeds of S. florida the difference between H202 treated and 4. 25°C constant 9. l1°C x 25 °C untreated seeds was non-significant (P= 0,44). Data for S. 5. 4°C x l1°C 10. 4°C X 25°C. florida were therefore pooled (Figure 1C) for the purpose of Alternating temperatures were obtained by transferring petri regression analysis. The dense fungal and bacterial growth on dishes of seeds from one to another of the four incubators seeds, observed in most petri dishes, may have contributed which were kept at constant temperatures. Seeds were incu­ to the difference in degree of reaction to H202 treatment of bated in petri dishes on a single sheet of Whatman no. 1 filter seeds germinated in petri dishes and in seed beds (Brits & Van paper which was kept damp with distilled water. A seed was Niekerk 1986). considered to have germinated when the radicle had emerged. The strong effect of diurnal temperature amplitude on Treatments were replicated three times with 50 seeds per germination percentage (Figure 1) suggests that seeds required replicate (petri dish) for L. cordifolium and 25 seeds per both low and high temperature to attain maximum germina­ replicate for S. florida. tion. It is therefore postulated that seeds of both species A viability test with tetrazolium (Brits & Van Niekerk 1976) require an optimum low (base) and an optimum high (ceiling) was carried out on L. cordifolium seeds, which indicated temperature for maximum germination. Regression analysis approximately 43% viability of these seeds. of data gave similar curved responses of germination in L. Mean daily temperatures of the regimes with alternating cordifo!ium and S. florida to varying mean daily temperatures temperatures were calculated using the following equation: and temperature amplitudes (Figure 2). Estimation of the Mean daily temperature = 2/ 3 (lower temperature) turning point (maximum) in each case allowed the calculation + 113 (higher temperature). of base and ceiling temperatures required for germination in The relationship between mean daily temperature, tempera­ the two species (Table 1). The high r2 values found indicate ture amplitude and germination percentage was determined close fits of regressions to data. by means of bivariate polynomial regression analysis. From It was assumed that L. cordifolium seeds in nature would these the turning points (maxima) were estimated of X1 (mean be covered only lightly by leaf litter or soil. This assumption daily temperature) and x2 (temperature amplitude) for the is supported by the known requirement of shallow sowing data of untreated L. cordifo!ium and the combined data of of seeds in seed beds to obtain maximum germination (V ogts S. florida seeds. Using the estimated turning points X1' and 1959). Surface soil temperature data for the natural habitat X2', the optimum low (base) and optimum high (ceiling) of L. cordifo!ium were not available. Available climatic data germination temperatures for the two species were calcu­ indicate, however, that the daily temperature regime at the lated by transposing the equation used to obtain mean daily experimental site (altitude 180m) and in the areas where L. temperatures: cordifolium grows naturally (median altitude 240 m) are Base temperature = X1' - 1/3X2' and reasonably similar. Ceiling temperature = X1' + 2/ 3X2'. Seeds of L. cordifolium germinate maximally during the five coolest months of May to September i.e. winter (Brits Determination of surface soil temperatures in burnt & Van Niekerk 1986). Mean daily minimum and maximum and unburnt tynbos surface soil temperatures and temperature amplitudes in burnt A 50 m2 area of mesic mountain fynbos, which had been and unburnt fynbos during this season and in summer are burnt 8 years previously, growing on shallow lithozolic soil given in Table 2. Although daily minima did not differ (mainly Cartref and Houwhoek Forms) on a south-facing, much, daily maxima and thus temperature amplitude were 13% slope of the Riviersonderend mountain on the Tygerhoek much higher in burnt exposed soil than in shaded soil. Of Experimental Farm, Riviersonderend (34°9'S, 19°54'E), was the four possible combinations of burnt/unburnt status divided into two blocks of equal size. One block was burnt of fynbos and winter/summer season only burnt fynbos in February 1983 and kept completely cleared of regenerating during winter gave daily minimum and maximum tempera­ vegetation during the f;Xperiment. In the adjacent unburnt tures (Table 2) which satisfy both the base and ceiling block two sites were arbitrarily chosen as representing average temperature requirements for maximum seed germination lightly shaded and average heavily shaded areas. Illumination (Table 1). Figure 3 illustrates the pattern of seasonal tempera­ at soil level at each of the three experimental sites in clear ture variations which probably would maintain seed dormancy 288 S.-Afr. Tydskr. Plantk., 1986, 52(4)

2 60 In Y = - 2,832 + 0,813X, - 0,029X1 A + 0,226X - 0,00767X,' s 50 2 'C Multiple r = 0,96 :; 40 .!2 4 c: -:; "iPO c: Gi 20 E (!) .. A ~ 0 10 ~ 2 0 ~ 2 c:

Temperature amplitude •c Temperature amplitude •c Mean temperature •c 11 18 temperature •c

2 60 3 In Y = -0,205 + 0,246X, - 0,0106X 1 8 + 0,311X, - 0,0116X2 .!~ 50 g ; 2 ~ 40 c: Multiple r = ~0~,9~0~H~~R~~ ·e 3o "§ ~ 20 ~ 1 ~ 10 0 21

Temperature amplitude •c Temperature amplitude •c

Mean temperature •c 5 Mean temperature •c Figure 2 Bivariate polynomial regressions of the data represented in Figure lA: Leucospermum cordifolium - untreated seeds (A) and c: 30 Figure IC: combined data of Serruria florida (B). Germination per­ .!2 c centages were transformed to natural logarithms to improve the fit of fti c: 20 data to regressions. "§ Cll (!) 10

~ 2 0 Table 1 Values of r , maximum X : mean hourly 0 1 2 temperature (X,'), maximum X2: temperature amplitude (X2'), optimum low (base) temperature, and optimum high (ceiling) temperature, estimated from regression Temperature for the data of untreated and treated Leucospermum amplitude °C cordifolium and the combined data of Serruria florida seeds

Mean temperature •c Species & Base Ceiling treatment r2 XI' X2' temp. ( 0 C) temp. (0 C) Figure 1 Three-dimensional bar diagrams of germination percentages found under 10 temperature regimes with different combinations of L. cordifo/ium mean daily temperatures and temperature amplitudes in seeds of A: (H20) 0,9225 14,03 14,76 9,11 23,87 Leucospermum cordifolium -untreated; B: L. cordifolium -treated L. cordifo/ium with I 07o H202; C: Serruria florida - combined data for untreated (H202) 0,9699 13,26 14,20 and treated seeds. Each bar represents the mean of three replications. S. florida 0,8159 11,54 13 ,42 7,07 20,48 partially in unbumt fynbos and would break dormancy during strongly with seedlings (Boucher 1981). This observation winter only in burnt fynbos. Since winter is also the season indicates that the effects of fire per se were not required for of high available soil moisture, L. cordifolium seeds will, there­ seed germination and supports the present hypothesis that, fore, germinate on a large scale during the first winter after fire. in exposed soil, germination is stimulated mainly by high day­ zeyheri Pappe ex Hook, a Proteaceae species temperature prevailing in the surface soil. with nut-like fruits, maintains viable seed banks in the soil Seed dormancy will not be broken in unburnt vegetation al­ for periods of up to 19 years (Boucher & McCann 1975; Van though the required base and ceiling temperatures may be met der Merwe 1977). Following hoeing of a locality containing in successive separate seasons of the year for an indefinite num­ only a few old moribund individuals, 0. zeyheri recruited ber of times (fable 2; Figure 3). Diurnal and not seasonal alter- S. Afr. J . Bot., 1986, 52(4) 289

Table 2 Mean daily minimum and maximum tempera­ lower average temperatures occur where S. florida grows tures and temperature amplitude (0 C) at 5 mm soil naturally, than in the case of L. cordifolium, thus supporting depth during the seven warmest (summer) and five the present finding. It seems, therefore, that close correlations coolest (winter: May to Sept.) months in burnt, unburnt: exist between ecological conditions and germination require­ lightly shaded, and unburnt: heavily shaded fynbos at ments in these two species. Tygerhoek Experimental Farm for the period April 1983 Low temperature requirement in germinating L. cordi­ to July 1984. Values close to estimated basea and folium seeds probably functions to avoid moisture stress ceilingb temperature requirements for maximum seed in new seedlings by maintaining dormancy during rainy, germination in L. cordifolium are indicated transiently favourable periods in the summer months (Brits & Van Niekerk 1986). On the other hand high temperature Seven warmest months Five coolest months requirement, causing the synchronous, en masse germination Light Heavy Light Heavy of seeds following fire, could function to avoid intra- and Burnt shade shade Burnt shade shade interspecific competition of seedlings (Angevine & Chabot Mean 1979) with both mature and rapidly regenerating fynbos maximum 45 ,7 35,6 25,5b 24,4b 17,9 15,8 vegetation (Kruger 1979; Vander Merwe 1966) during their Mean establishment phase. minimum 13,7 15,2 15,6 6,3• 8,8• 9,1• Genera of Proteaceae with nut-like fruits share a number Temp. of seed morphological and dispersal characteristics (Vogts amplitude 32,0 20,4 9,9 18,1 9,1 6,7 1982). The two species of different genera in the present study have similar thermoperiodic germination requirements (Figure 2). These requirements are therefore probably common adaptations in Proteaceae with nut-like fruits to two stress factors in fynbos viz. summer drought and cyclical fires. The model proposed above does not apply to serotinous species of Proteaceae. The seeds of these species (e.g. ) are held in fire-resistant infructescences in the plant canopy until the effects of fire cause them to be released (Bond 1984). This implies that seed dormancy in nature would be advanta­ geous only during the warm season following dispersal, which preceeds the first winter after frre (Deall & Brown 1981; Bond O~A-LM~-J~~~A~S~-O~~N~7DJ_J~~~M-LA~~M~~~ 1984). When stratified, seeds of Link ap­ (1983) MONTH (1984) parently required only low temperature for germination (Deall 0 ·Maximum temperatures ) t & Brown 1981). Seeds of serotinous Proteaceae species are • Minimum temperatures burn exposed 6 Maximum temperatures l unburnt: therefore not quiescent (Villiers 1972; Jann & Amen 1977) • Minimum temperatures f lightly shaded as suggested by Deall and Brown (1981) but dormant by 0 Maximum temperatures ) unburnt: e Minimum temperatures heavily shaded means of low temperature requirement (Bond 1984). The annual seed dispersal in species with nut-like fruits, however, Figure 3 Mean daily maximum and minimum temperatures for necessitates an additional dormancy mechanism by means of individual months measured at 5 mm soil depth in burnt, unbumt: lightly high temperature requirement, in order to recognize the early shaded, and unburnt: heavily shaded (see Materials and Methods) fynbos of 8 years post-fire age for the period April 1983 to July 1984 at post-fire situation. Tygerhoek Experimental Farm, Riviersonderend. Temperatures were recorded during five days per week. The two broken lines indicate the Acknowledgements estimated low and high temperature requirements in Leucospermum The assistance of the following is gratefully acknowledged: cordijolium. Department of Biometry and Datametrical Services of the Fruit and Fruit Technology Research Institute, Stellenbosch; nation of base and ceiling temperatures are therefore required Jonkershoek Forestry Research Centre, Stellenbosch; Section for germination. This suggests that different physiological for Meteorology of the Winter Rainfall Region. processes, possibly associated with low and high temperature requirements, must occur concomitantly to initiate germina­ References tion. This proposal is consistent with the hypothesis that the onset of germination in daphnoides (Thunb.) ANGEVINE, M.W. & CHABOT, B.F. 1979. Seed germination syndromes in higher plants. In: Topics in plant population Meisn., another Proteaceae species with nut-like fruits, involves biology, eds Solbrig, O.T., Jain, S., Johnson, G.B. & Raven, two simultaneous (non-phasic) physiological processes (Brown P.H., Ch. 8, Columbia University Press, New York. & Van Staden 1975). It also supports the view that the daily BENIC, L.M. & KNOX-DAVIES, P.S. 1983. Anthracnose of low temperature requirement during germination in these seeds , caused by Colletotrichum gloeosporioides. is not a stratification requirement (Brits & Van Niekerk 1986) Phytophylactica 15: 109-119. BOND, W.J. 1984. Fire survival of Cape Proteaceae - influence since the latter is characteristically a prolonged separate phase of fire season and seed predators. Vegetatio 56: 65- 74. of after-ripening at sub-optimal temperature, during which BOUCHER, C. 1981. Autecological and population studies of germination does not occur (Lewak & Rudnicki 1971). Orothamnus zeyheri in the Cape of South Afriq. In: The In spite of similar germination patterns, substantially lower biological aspects of rare plant conservation, ed. Synge, H., base and ceiling temperatures were calculated for S. florida Ch. 28, John Wiley & Sons Ltd. than for L. cordifoliurh seeds (Table 1). Known germination BOUCHER, C. & McCANN, G. 1975. The Orothamnus saga. Veld & Flora 61: 2 - 5. requirements when in cultivation (Vogts 1959), a description BRITS, G.J . & VAN NIEKERK, M.N. 1976. Opheffing van of the habitat (V ogts 1982) which is located at a higher altitude saadrus by Leucospermum cordijolium (Proteaceae). (500 m), and limited available climatic data, all indicate that Agroplantae 8: 91-94. 290 S.-Afr. Tydskr. Plantk., 1986, 52(4)

BRITS, G.J. & VAN NIEKERK, M.N. 1986. Effects of air dormancy and germination, ed. Khan, A.A., Ch. 10, North temperature, oxygenating treatments and low storage Holland Publishing Company, Amsterdam. temperature on seasonal germination response of ROURKE, J.P. 1972. Taxonomic studies on Leucospermum R.Br. Leucospermum cordijolium (Proteaceae) seeds. S. Ajr. J. Bot. Jl S. Afr. Bot. Supplementary volume No. 8, Trustees of the 52: 207-211. National Botanic Gardens of South Africa, Kirstenbosch, BROWN, N.A.C. & DIX, L. 1985. Germination of fruits of Newlands, C.P. Leucadendron tinctum. S. Afr. J. Bot. 51: 448-452. SNEDECOR, G.W. & COCHRAN, W.G. 1967. Statistical BROWN, N.A.C. & VAN STADEN, J. 1971. Germination Methods, 6th edn, The Iowa State University Press, Ames, inhibitors in aqueous seed extracts of four South African Iowa. Proteaceae. Jl S. Afr. Bot. 37: 305-315. THOMPSON, P.A. 1974. Effects of fluctuating temperatures on BROWN, N.A.C. & VAN STADEN, J. 1973. The effect of germination. J. exp. Bot. 25: 164 - 175 . scarification, leaching, light, stratification, oxygen and applied VANDER MERWE, P . 1966. Die flora van die Swartboskloof, hormones on germination of Protea compacta R.Br. and Stellenbosch en die herstel van die soorte na 'n brand. Annale Leucadendron daphnoides Meisn. Jl S. Ajr. Bot. 39: 185-195. Universiteit van Stellenbosch, Vol. 41 Series A No. 14. DEALL, G.B. & BROWN, N.A.C. 1981. Seed germination in VAN DER MER WE, P. 1977. Die morfologie en voortplanting Protea magnifica Link. S. Ajr. J. Sci. 77: 175-176. van Orothamnus zeyheri (Proteaceae). Research Report: Plants. HORN, W. 1962. Breeding research on South African plants. II. 1977, Department of Nature and Environmental Conservation, Fertility of Proteaceae. Jl S. Ajr. Bot. 28: 259- 268. Provincial Administration of the Cape of Good Hope. JANN, R.C. & AMEN, R.D. 1977. What is germination? In: The VAZQUES-YANES, C. & OROZCO-SEGOVIA, A. 1982. Seed physiology and biochemistry of seed dormancy and germination of a tropical rain forest pioneer tree (Heliocarpus germination, ed. Khan, A.A., Ch. 2, North Holland Publishing donnell-smithit) in response to diurnal fluctuation of Company, Amsterdam. temperature. Physiologia Pl. 56: 295 - 298 . KOLLER, D. 1972. Environmental control of seed germination. VILLIERS, T.A. 1972. Seed dormancy. In: Seed Biology II. In: Seed biology II. Physiological ecology, ed. Kozlowski, T.T., Physiological ecology, ed. Kozlowski, T.T., Ch. 3, Academic Ch. I, Academic Press, New York. Press, New York. KRUGER, F.J. 1979. Fire. In: Fynbos ecology: a preliminary VOGTS, M.M. 1959. - know them and grow them, Ch . synthesis, eds Day, J., Siegfried, W.R., Louw, G.N. & 4, Afrikaanse Pers-Boekhandel (Edms.) Bpk., Johannesburg. Jarman, M.L. South African National Scientific Programmes VOGTS, M.M. 1976. Species and variants of proteas. Fmg. S. Report No. 40, printed by the Graphic Arts Division of the Afr. Leaflet Series. Flowers, ornamental and trees B.!, CSIR, Pretoria. Government Printer, Pretoria. LEWAK, S. & RUDNICKI, R.M. 1977. After-ripening in cold­ VOGTS, M.M. 1982. South Africa's Proteaceae - know them requiring seeds. In: The physiology and biochemistry of seed and grow them, C. Struik (Pty) Ltd, Cape Town.