Seed storage behaviour and seed germination in ...

Seed Science Research (2006) 16, 83 – 88

DOI: 10.1079/SSR2005231

SHORT COMMUNICATION

Seed storage behaviour and seed germination in African and Malagasy baobabs (Adansonia species) Juvet Razanameharizaka1, Michel Grouzis2, Didier Ravelomanana1 and Pascal Danthu3* 1

Universite´ d’Antananarivo, Laboratoire de Physiologie Ve´ge´tale / URP 70 ‘Foreˆts et Biodiversite´’, B.P. 906, Antananarivo, Madagascar; 2IRD, B.P. 434, Antananarivo, Madagascar; 3Cirad / URP 70 ‘Foreˆts et Biodiversite´’, B.P. 853, Antananarivo, Madagascar

Abstract

Introduction

The Adansonia (baobab) genus comprises seven species in Africa, six of which are endemic to Madagascar. Depending on the species, baobabs develop in widely varying ecosystems, including arid zones and savannahs, as well as dry and wet forests. Seeds from all species exhibited orthodox behaviour, tolerating dehydration to a moisture content of around 5%. There was no physical dormancy in the two species belonging to the Brevitubae section, A. grandidieri and A. suarezensis. Their seeds germinated without any prior scarification. The five other species, belonging to Adansonia and Longitubae section, have seeds with water-impermeable coats. In the case of A. digitata and A. za, the proportion of water-impermeable seeds was around two-thirds, whereas with A. rubrostipa, A. madagascariensis and A. perrieri, the proportion was . 90%. Treatments allowing for the removal of physical dormancy needed to be markedly more severe with A. madagascariensis than with the other species. None the less, it seems impossible to link these characteristics and the interspecific differences to a strategy for adaptation by these species to their environment.

The Adansonia genus (family of Bombacaceae belonging to the Malvaceae family, sensu lato) is represented worldwide by eight species divided into three sections: Brevitubae, Adansonia and Longitubae (Table 1), which are distinguished primarily by the morphology of their flower buds and the lengths of the staminal tube (Jumelle and Perrier de la Baˆthie, 1912; Perrier de la Baˆthie and Hochreutiner, 1955; Baum, 1995, 1996, 2003; Schatz, 2001). These species grow in broadly different ecosystems. A. gibbosa originates in north-eastern Australia (Baum, 1995), whereas the distribution of A. digitata corresponds to the savannah zone of continental Africa (Owen, 1974; Wickens, 1982). This species was introduced into Madagascar during the past few centuries (Wickens, 1982). The six other species are all endemic to the Malagasy forests (Perrier de la Baˆthie and Hochreutiner, 1955; Baum, 1995). A. rubrostipa is dependent on the thickets and dry forests of the west coast. A. grandidieri is found in two locations of the western dry forest, one close to Morombe and the other to the north of Morondava. A. perrieri, which has a more restricted distribution, is constrained to the evergreen wet forests of Montagne d’Ambre, while A. suarezensis is only present in the deciduous forests close to Diego Suarez (Antsiranana). A. madagascaiensis develops in the dry and wet forests between Mahajanga and Antsiranana. Finally, A. za enjoys the widest distribution, extending from the spiny bush in the Tolagnaro (Fort Dauphin) region to the evergreen forest of Sambirano (Baum, 1995, 1996). The objective of this study was to clarify a few aspects of the conservation and germination of baobab seeds, concentrating ultimately on determining the status of seeds (orthodox versus recalcitrant), and

Keywords: Adansonia, Africa, baobab, germination, Madagascar, seed storage behaviour

*Correspondence Fax: þ261 20 22 408 21 Email: [email protected]

J. Razanameharizaka et al.

36 27 33 27 30 30

4.7 ^ 0.9 ND 7.7 ^ 0.5 ND 5.9 ^ 0.8 5.1 ^ 0.6

assessing physical dormancy in the different species. The suggested protocol consists in comparing the imbibition of seeds from different Adansonia species, and their germination before and after seed coat scarification of increasing intensities. Three previously adopted methods for removing seed coat impermeability in A. digitata were utilized: manual scarification, soaking in a concentrated sulphuric acid solution and scalding the seeds (Booth and Wickens, 1988; Esenowo, 1991; Danthu et al., 1995). The differences revealed between species are discussed in the light of known ecological and/or taxonomic aspects. Materials and methods

ND, not determined; fwb, fresh weight basis.

A. madagascariensis Baill. A. perrieri Capuron

A. za Baill.

Longitubae A. rubrostipa Jum & H. Perrier

Sample Sample Sample Sample Sample Sample

1: 2: 1: 2: 1: 1:

Ifaty (SW Madagascar) 238040 S – 438370 E Kirindy (W. Madagascar) 208040 S – 448300 E Analabo (SW Madagascar) 228310 S– 438310 E Kirindy (W. Madagascar) 208040 S – 448300 E Anjiamangirana (NW Madagascar) 158110 S– 478500 E Foreˆt d’Ambre (N. Madagascar) 128040 S – 498170 E

719 ^ 19 759 ^ 48 568 ^ 34 495 ^ 31 366 ^ 9 710 ^ 6

13.8 ^ 0.2 13.4 ^ 0.3 13.5 ^ 0.2 11.3 ^ 0.3 10.9 ^ 0.4 12.9 ^ 0.2

6.1 ^ 0.6 ND 28 27 397 ^ 8 427 ^ 14

A. suarezensis H. Perrier Adansonia A. digitata L.

Sample 1: Bandia (W. Senegal) 148340 N – 178010 W Sample 2: Mahajanga (W. Madagascar) 158430 N – 468190 E

11.3 ^ 0.4 10.7 ^ 0.1

4.7 ^ 0.7 ND 4.1 ^ 0.5 40 8 12 15.8 ^ 0.4 14.9 ^ 0.3 16.9 ^ 0.5 803 ^ 33 1053 ^ 62 1717 ^ 48 Brevitubaea A. grandidieri Baill.

Origin of seeds

Sample 1: Tandila (W. Madagascar) 208100 S – 448250 E Sample 2: Morondava (W. Madagascar) 208150 S –448190 E Sample 1: Behantely (N. Madagascar) 128160 S – 498170 E

Age of seeds from harvest (months) Section and species

Table 1. Origin and biometric characteristics of Adansonia seeds used in this study

Weight of seeds (mg)

Length of seeds (mm)

Moisture content of seeds at harvest (% fwb)

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This study focuses on the seven species of baobab present in Africa and Madagascar. Ripe fruits were collected in the natural habitat of each species: in Senegal for A. digitata and Madagascar for all species (Table 1). After fruit collection, the seeds were extracted from the pulp and stored in a dry atmosphere at room temperature (15/258C) until use. Four experiments were carried out. The objective in the first was to test the hypothesis that germination was inhibited by the seed coat. The experiment compared the germination capacity of intact seeds with that of seeds from which a 5–10 mm2 fragment of coat had been removed with pruning shears. The second experiment consisted in comparing the impermeability of the seed coats from different species. It was performed on one or two batches per species. In this experiment the moisture content of five replicates 3– 5 seeds from each of the species was measured before and after soaking in water at 30 ^ 18C for 12 h. The third and fourth experiments were reserved for the species that had demonstrated water-impermeable seed coats in the two first experiments. The objective was to compare different treatments that allow for the removal of physical dormancy. The third experiment involved assessing the duration of soaking in concentrated sulphuric acid (H2SO4, 95%): 30 min, 1 h, 3 h, 6 h and 12 h (followed by abundant rinsing under running water). The fourth experiment consisted in assessing the duration of immersion in a large volume (around 2 litres) of boiling water: 15, 60 and 180 s. Biometric parameters (weight and length of seeds) were measured on a sample of 30 seeds. Seed moisture contents were measured on five replicates of 3– 5 coarsely crushed seeds that were initially weighed and then heated at 1058C until weight stabilization. The moisture content was expressed on the fresh weight basis (fwb) (ISTA, 1999). Germination tests were carried out according to ISTA (1999) directives using plastic germination boxes

Seed storage behaviour and seed germination in Adansonia species (17 £ 11 £ 5 cm), in a dark confined atmosphere, on a bed of sterilized sand moistened with distilled water at 30 ^ 18C. A seed was considered germinated if the radicle emerged through the seed coat. At the end of the experiment, the viability of ungerminated seeds was estimated by dissection. The results were expressed as germination capacity (percentage of seeds germinated 25 d after sowing), or relative germination (RG), which allows for comparison between species. In this case, RG [adapted from Sharma (1973)] corresponds to the ratio of germination capacity in a given experimental condition to the germination capacity obtained after manual scarification, a condition that makes it possible to come closest to the germination power of the seed lots under consideration (Danthu et al., 2000). Each experiment was performed on four replicates of 50 seeds. Analyses of variance were carried out after using an angular transformation of the percentages. When the F value was significant (P , 0.05), means were compared with the Newman – Keuls test at the threshold of P , 0.05. The means of biometric measurements (weight, length and moisture content of seeds) are given ^ the standard deviation at P , 0.05. Comparison of means in pairs was performed using the Student’s t-test with P , 0.05.

Results Seeds from all species have high germination capacity, between 86 and 100%, after manual scarification (Table 2). Seeds of A. grandidieri and A. suarezensis achieved 94 –96% germination without any pre-treatment, and this result was not signifiTable 2. Germination capacity (measured after 25 d) of intact Adansonia seeds and seeds scarified by removal of a small piece of seed coat (Newman –Keuls test at P , 0.05 applied to each row). Relative germination of intact seeds is given in order to compare species (Newman –Keuls test at P , 0.05 applied to the column, values with the same letter were not significantly different) Germination capacity (%) Species (sample 1 for all species) A. grandidieri A. suarezensis A. digitata A. rubrostipa A. za A. madagascariensis A. perrieri

Intact seeds

After manual scarification

Relavtive germination of intact seeds

96 a 94 a 27 b 7b 28 b 3b 8b

98 a 100 a 99 a 100 a 86 a 98 a 96 a

0.98 A 0.94 A 0.27 B 0.07 C 0.33 B 0.03 C 0.08 C

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cantly improved by scarification. Germination capacity for intact seeds of the five remaining species proved weak: 3–8% for A. madagascariensis, A. rubrostipa, A. perrieri and 27–28% for A. digitata and A. za; germination was significantly higher (96– 100%) when the seeds were scarified before sowing. Comparing relative germination of intact seeds produces three groups: (1) A. grandidieri and A. suarezensis produced a RG . 0.94, indicating that all viable seeds germinated without pre-treatment; (2) A. digitata and A. za with 0.27 # RG # 0.33, signifying that around two-thirds of viable seeds were water impermeable; and (3) A. rubrostipa, A. madagascariensis and A. perrieri for which an RG , 0.1 indicated that more than 90% of seeds have impermeable coats. For seeds of A. digitata, A. za, A. rubrostipa, A. madagascariensis and A. perrieri, the moisture content was not significantly modified by soaking for 12 h in water. However, for seeds of A. grandidieri and A. suarezensis, a net increase in moisture content was observed, rising from 5–7% to 15 and 23%, respectively (Table 3). For A. grandidieri, A. digitata, A. rubrostipa and A. za, two batches differing in origin and ages were tested. Similar behaviour was observed in each batch. The maximum moisture content values obtained after soaking for A. za and A. perrieri were as much as 13%. The following experiments (Figs 1 and 2) concern the five species exhibiting water-impermeable seed coats. Figure 1 compares the relative germination of seeds after soaking in concentrated sulphuric acid for progressively increasing durations. For A. madagascariensis, optimal germination (RG . 0.95) was obtained only when seeds were acid-scarified for a minimum of 12 h. For the other four species (A. digitata, A. rubrostipa, A. za and A. perrieri), RG . 0.95 was attained with only 3 h of acid soaking. Differences could still be detected between these four species: germination after 1 h of soaking was significantly higher for A. digitata (RG ¼ 0.76) and A. za (RG ¼ 0.61), relative to A. rubrostipa and A. perrieri (RG , 0.30). In all of the experimental conditions, for all these species, seeds remaining ungerminated at the end of the experiment were still viable, indicating that the treatments had not eliminated seed coat impermeability. Scalding with water also significantly improved germination of the baobab seeds, if the treatment was applied for 15 s to A. digitata, A. perrieri and A. za, for 15 –60 s to A. rubrostipa and 60 s in A. madagascariensis (RG $ 0.89). Beyond these durations, germination capacities dropped significantly, in particular in A. perrieri, for which germination was nil if the soaking exceeded 1 min. For all these species, seeds remaining ungerminated at the end of the experiment were necrotic, indicating that the treatments applied were lethal.


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Table 3. Moisture content (relative to the weight of fresh material) of Adansonia seeds after 12 h soaking in water; the mean is given bracketed by the two extreme values [comparison of means in pairs using Student’s t-test; means are said to be significantly different if P , 0.05 (t . 2.306 with 8 df) and are marked *]. Moisture content of seeds (% fwb) Species A. grandidieri A. suarezensis A. digitata A. rubrostipa A. za A. madagascariensis A. perrieri

Sample

Control (intact seeds)

After soaking in water (12 h)

P value

1 2 1 1 2 1 2 1 2 1 1

5.1 , 5.5 . 5.8 6.3 , 7.3 . 7.7 4.4 , 5.2 . 6.0 5.6 , 6.1 . 6.5 7.1 , 7.5 . 8.0 5.8 , 6.0 . 6.5 4.8 , 6.6 . 8.2 6.0 , 6.3 . 6.6 7.0 , 7.7 . 8.7 5.9 , 6.4 . 7.4 7.0 , 7.3 . 7.5

13.0 , 15.9 . 18.0 13.6 , 15.6 . 18.6 17.5 , 23.0 . 28.8 5.0 , 6.3 . 7.7 6.7 , 8.4 . 9.9 6.2 , 7.0 . 8.4 4.4 , 6.5 . 8.1 6.2 , 8.6 . 13.1 7.4 , 7.5 . 7.8 6.3 , 6.8 . 7.2 7.5 , 8.7 . 12.8

, 0.0001* , 0.0001* , 0.0001* 0.725 0.246 0.052 0.891 0.104 0.482 0.200 0.203

fwb, fresh weight basis.

Discussion The seeds from the seven baobab species studied have a harvest moisture content between 4 and 8% (relative to the weight of fresh material) (Table 1), and the germination capacity is high (86% for A. za and approaching 100% for all the other species) (Table 2). This information allows these baobab species to be classified as orthodox seeds (Roberts, 1973; Bonner, 1990). For the first group of five species (A. digitata, A. rubrostipa, A. za, A. madagascariensis and A. perrieri), the removal of a fragment of seed coat significantly increased their capacity to germinate (Table 2). These five species possess seeds with physical dormancy. 1.0

However, a fraction of their seeds had the capacity to germinate without scarification: around a third of seeds from A. digitata and A. za and less than 10% of seeds from the other species, which explains the evidence of a few cases of imbibition in A. za, and A. perrieri in the second experiment (Table 3). A. grandidieri and A. suarezensis constitute a second group of baobabs, the seeds of which have the capacity to germinate without scarification before sowing (Table 2).The seeds from these two species are water permeable (Table 3). The results obtained from the first group confirm the conclusions of Esenowo (1991) and Danthu et al. (1995) concerning A. digitata. They also correspond to the generally acknowledged observation that species

a

0.8

b c

0.6 d

0.4

e 0.2 f 0.0

0

1

A. digitata A. rubrostipa A. za A. madagascariensis A. perrieri

3 6 Duration of soaking in sulphuric acid (h)

12

Figure 1. Relative germination of seeds from five Adansonia species according to the time of soaking in concentrated sulphuric acid. Vertical bars with the same letter indicate homogeneous groups as defined by Newman– Keuls test at P , 0.05 applied to all values.

Relative germination

Relative germination

1.0

a

0.8 0.6

A. digitata A. rubrostipa A. za A. madagascariensis A. perrieri

b

0.4 c 0.2 d 0.0

0

15

60 Duration of soaking in boiling water (s)

180

Figure 2. Relative germination of seeds from five Adansonia species according to the time of soaking in boiling water. Vertical bars with the same letter indicate homogeneous groups as defined by Newman– Keuls test at P , 0.05 applied to all values.

Seed storage behaviour and seed germination in Adansonia species of the Malvaceae family have seeds with impermeable coats (Ballard, 1973; Rolston, 1978; Egley and Paul, 1981). On the other hand, seeds of A. grandidieri and A. suarezensis do not have physical dormancy. This observation does not appear to be associated with storage time, as observed for Sida spinosa (Egley and Paul, 1982); the two batches of A. grandidieri seeds from different harvest years had the same behaviour. Moreover, it would be reasonable to exclude the hypothesis of a bias due to a batch of seeds having been harvested before full maturity, because all the batches had the same behaviour. The first conclusion to be drawn from our results is that the baobab species belonging to the Adansonia and Longitubae sections generally have seed coats that are water impermeable, while those from the Brevitubae section have seeds without this physical barrier (Tables 1, 2). The five species presenting impermeable seed coats do not manifest identical behaviour. Seeds from A. digitata, A. za, A. rubrostipa and A. perrieri germinated entirely after scarification by soaking in concentrated sulphuric acid for periods ranging from 3 to 12 h or by boiling for 15 s (Figs 1, 2). This behaviour resembles that observed for A. digitata by Danthu et al. (1995). The fact that A. digitata and A. za germinated better than A. perrieri and A. rubrostipa, after a relatively short time soaking in acid (an hour for example), is explained essentially by the fact that the first two species possess a lower proportion of seeds with physical dormancy (70%) compared to the latter two species (90%). The behaviour of seeds from A. madgascariensis differed significantly compared to the other four species. This species required the most drastic treatment in order to germinate: a minimum of 12 h soaking in sulphuric acid and scalding for 1 min (Figs 1, 2). Thus, it seems possible to classify the baobab species into three groups based on the water impermeability of their seeds: (1) species for which the seeds have permeable coats: A. grandidieri and A. suarezensis; (2) species with significant physical dormancy: A. digitata, A. za, A. perrieri and A. rubrostipa; and (3) species with the greatest physical dormancy: A. madagascariensis. However, it is difficult to correlate these differences with their ecological characteristics. Hence, A. grandidieri and A. suarezensis inhabit very distinct ecosystems (Baum, 1995), but have identical behaviour. Conversely, A. madagascariensis and A. perrieri, which develop in the same ecosystems, manifest stark differences in the physiology of their germination. A. rubrostipa, which is the most xerophytic of the species, is not the one that presents the greatest seed coat water impermeability, and yet this feature is often associated with adaptation to arid environments (Rolston, 1978; Gutterman, 1994). Likewise, A. madagascariensis seeds, which disperse through hydrochory (Baum, 1995; Du Puy,

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1996), have the greatest seed coat impermeability in relation to A. digitata, for which zoochory is the recognized means of dispersal (Owen, 1974; Wickens, 1982), despite the fact that the survival of seeds during the passage through the digestive tract of animals is generally associated with seed coat impermeability (Gardener et al., 1993). This study indicates that seeds from African and Malagasy baobab are orthodox. On the other hand, they present a wide range of physical dormancy; certain species appear not to develop any impermeability, while others manifest substantial impermeability unconnected to any ecological or adaptive considerations. However, as demonstrated by Danthu et al. (1995), this result can depend on the seed lots used. It would be useful to verify the results on different lots from each species. Moreover, removal of physical dormancy does not require scarification of the entire seed coat, but only in a weak spot. In the case of Malvaceae, this corresponds to the chalaza (Ballard, 1973; Egley and Paul, 1981; Serrato-Valenti et al., 1992). A histological study of the seed coats from the different species, and in particular of the chalazal region, is in progress to attempt to expose tissue differences that might explain the physiological responses uncovered in this study. Acknowledgements This study was conducted by the URP ‘Foreˆts et Biodiversite´’, a collaboration between the University of Antananarivo, Fofifa and Cirad, as part of a research programme, within the Projet Corus No. 02 518 317 145. It received funding from the French Ministry of Foreign Affairs (projects FSP/Forma and FSP/GDRN). The authors wish to thank the SNGF (Silo National de Graines Forestie`res) for its technical support. References Ballard, L.A.T. (1973) Physical barriers to germination. Seed Science and Technology 1, 285 –303. Baum, D.A. (1995) A systematic revision of Adansonia (Bombacaceae). Annals of Missouri Botanical Garden 82, 440–470. Baum, D.A. (1996) The ecology and conservation of the baobabs of Madagascar. Primate Report 46, 311– 327. Baum, D.A. (2003) Bombacaceae, Adansonia, Baobab, Bozy, Fony, Renala, Ringy, Za. pp. 339– 342 in Goodman, S.M.; Benstead, J.P. (Eds) The natural history of Madagascar. Chicago, University of Chicago Press. Bonner, F.T. (1990) Storage of seeds: potential and limitations for germplasm conservation. Forest Ecology and Management 35, 35– 43. Booth, F.E.M. and Wickens, G.E. (1988) Non-timber uses of selected arid zone trees and shrubs in Africa. FAO

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conservation guide 19. Rome, Food and Agriculture Organization of the United Nations. Danthu, P., Roussel, J., Gaye, A. and El Mazzoudi, E.H. (1995) Baobab (Adansonia digitata L.) seed pretreatments for germination improvement. Seed Science and Technology 23, 469– 475. Danthu, P., Gue`ye, A., Boye, A., Bauwens, D. and Sarr, A. (2000) Seed storage behaviour of four Sahelian and Sudanian tree species (Boscia senegalensis, Butyrospermum parkii, Cordyla pinnata and Saba senegalensis). Seed Science Research 10, 183– 187. Du Puy, B. (1996) The baobabs of Madagascar. Curtis’s Botanical Magazine 13, 86 –95. Egley, G.H. and Paul, R.N. (1981) Morphological observations on the early imbibition of water by Sida spinosa (Malvaceae) seed. American Journal of Botany 68, 1056 – 1065. Egley, G.H. and Paul, R.N. (1982) Development, structure and function of subpalisade cells in water impermeable Sida spinosa seeds. American Journal of Botany 69, 1402 – 1409. Esenowo, G.J. (1991) Studies on germination of Adansonia digitata seeds. Journal of Agricultural Science 117, 81– 84. Gardener, C.J., McIvor, J.G. and Jansen, A. (1993) Passage of legume and grass seeds through the digestive tract of cattle and their survival in faeces. Journal of Applied Ecology 30, 63 –74. Gutterman, Y. (1994) Strategies of seed dispersal and germination in plant inhabiting deserts. Botanical Review 60, 373– 425. ISTA (International Seed Testing Association) (1999) International rules of seed testing. Seed Science and Technology 27 (supplement 1).

Jumelle, H. and Perrier de la Baˆthie, H. (1912) Les baobabs du Sud-Ouest de Madagascar. Revue Geńe´ rale de Botanique 24, 372– 380. Owen, J. (1974) A contribution to the ecology of the African baobab (Adansonia digitata L.). Savanna 3, 1 – 12. Perrier de la Baˆthie, H. and Hochreutiner, B.P.G. (1955) Bombacaceés (Bombacaceae). pp. 1– 20 in Humbert, H. (Ed.) Flore de Madagascar et des Comores, 129e`me famille et 130e`me famille. Paris, Gouvernement Geńe´ral de Madagascar. Roberts, E.H. (1973) Predicting the storage life of seeds. Seed Science and Technology 1, 499– 514. Rolston, M.P. (1978) Water impermeable seed dormancy. Botanical Review 44, 365– 396. Schatz, G.E. (2001) Malvaceae Juss. pp. 253– 269 in Generic tree flora of Madagascar. Kew, Royal Botanic Gardens and St. Louis, Missouri Botanical Garden Press. Serrato-Valenti, G., Cornara, L., Lotito, S. and Quagliotti, L. (1992) Seed coat structure and histochemistry of Abelmoschus esculentus. Chalazal region and water entry. Annals of Botany 69, 313– 321. Sharma, M.L. (1973) Simulation of drought and its effect on germination of five pasture species. Agronomy Journal 65, 982– 987. Wickens, G.E. (1982) The baobab - Africa’s upside-down tree. Kew Bulletin 37, 173 –209.

Received 18 February 2005 accepted after revision 10 November 2005 q CAB International 2006