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Germination and emergence of the hard seed coated Tylosema esculentum (Burch) A. Schreib in response to different pre-sowing seed treatments
ARTICLE IN PRESS Journal of Arid Environments
Journal of Arid Environments 68 (2007) 501–507 www.elsevier.com/locate/jnlabr/yjare
Short communication
Germination and emergence of the hard seed coated Tylosema esculentum (Burch) A. Schreib in response to different pre-sowing seed treatments I.S. Travlos, G. Economou, A.I. Karamanos Agricultural University of Athens, Department of Crop Production, Laboratory of Agronomy, 75, Iera Odos st., 11855 Athens, Greece Received 17 November 2005; received in revised form 18 May 2006; accepted 3 July 2006 Available online 15 August 2006
Abstract Marama bean (Tylosema esculentum (Burch) L. Schreib) is an underutilized drought-tolerant legume native to the arid and semi-arid regions of southern Africa, which produces protein- and oilrich seed and tubers with potential for use as human food and animal fodder. Laboratory experiments were conducted in Athens, in order to investigate germination behaviour of untreated seeds and seeds subjected to different dormancy-breaking treatments and subsequent emergence of the seedlings. The effect of some scarification methods on seed germination was estimated at 30 1C. Most treatments, likewise control, resulted to relatively high germination and emergence percentages, confirming that marama seeds have no physiological dormancy. The speed and percentage of T. esculentum seed germination was greatly increased by mechanical massive scarification of the seeds with sandpaper. These highly positive responses of marama seeds to the mechanical treatment clearly indicate that there is a moderate coat-imposed dormancy in this species. Immersion in water for 20 h and in concentrated sulphuric acid for 20 min were also some of the most effective treatments. r 2006 Elsevier Ltd. All rights reserved. Keywords: Seed dormancy; Hard seed coat; Mechanical scarification; Marama; Africa
Corresponding author. Tel./fax:+3 210 529 4482.
E-mail addresses: [email protected] (I.S. Travlos), [email protected] (G. Economou), [email protected] (A.I. Karamanos). 0140-1963/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2006.07.001
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1. Introduction Marama bean (Tylosema esculentum (Burch.) A. Schreib) is a wild perennial legume, indigenous to the arid and semi-arid grasslands of southern Africa (Bousquet, 1981-1982). The remarkable socio-economic value of the plant in African communities is mainly explained by the fact that the bean is highly nutritious when cooked (Keegan and Vanstaden, 1981), with protein and oil content comparable to soybean (Glycine max) and groundnut (Arachis hypogaea), respectively (Bower et al., 1988; Ketshajwang et al., 1998). Because of its great ability to survive under unfavourable conditions it can be considered suitable for cultivation, especially under preventive conditions for other crops (National Academy of Sciences, 1979; Keegan and Vanstaden, 1981). Marama seeds are relatively large, with a hard seed coat, able to resist mechanical damage, likewise seeds of other perennial legume species (Hyde, 1954). In the nature, there are various biotic and abiotic factors that promote seed scarification, such as mechanical abrasion of the seed coat by sand and rocks in water courses (Vilela and Ravetta, 2001). Under laboratory conditions and in agriculture, other methods to render permeable hard seed coats have been adopted and discussed in detail by Cavanagh (1987). The objective of this study was to assess the influence of several seed scarification methods on germination and emergence of T. esculentum. The potential establishment of this species as a crop and the risk of its over-exploitation, with the attendant loss of biodiversity (seed is gathered from the wild and widely consumed by local people and range animals) make imperative the need of such a study, in order to optimize its seed germination and first growth.
2. Materials and methods T. esculentum seeds were collected directly from the wild in Letlhakane, Botswana (211 340 S; 251 420 E) and stored at 4 1C and 50% RH until their use 10 months later (fresh weight was ranged between 2 and 3 g). Two germination experiments were carried out in completely randomized designs in incubators (Conviron T 38/Lb/AP) at constant temperature (30 1C) and total darkness. In the first experiment, the effects of the following treatments on seed germination were evaluated: (1) immersion in water at room temperature (25–30 1C) for 20 h; (2) immersion in concentrated (95%) sulphuric acid for 20 min followed by thorough rinsing with running water; (3) immersion in water for 10 h followed by immersion in concentrated sulphuric acid for 20 min and thorough rinsing with running water; (4) immersion in a gibberellic acid (GA3) solution (100 mg GA3 l1 water) for 40 min and (5) immersion in water for 10 h and in the above-mentioned GA3 aqueous solution for 40 min successively. In the second germination assay, seeds were subjected to (1) immersion in concentrated (95%) sulphuric acid for 20 min followed by thorough rinsing with running water; (2) immersion in concentrated (95%) sulphuric acid for 40 min followed by thorough rinsing with running water; (3) immersion in a gibberellic acid (GA3) solution (100 mg GA3 l1 water) for 40 min and (4) mechanical scarification (massive abrasion of the seeds between two sheets of sandpaper). Eight replicates (Petri dishes) were used for each treatment. Five seeds were placed between two Whatman no. 1 paper filter disks (Whatman Ltd., Maidstone, England) in
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each 9-cm Petri dish, and 5 ml of distilled water was added whenever there was a need to keep filter papers moist. Seed germination was recorded every other day and expressed as a percentage of the total number of tested seeds (germination percentage, GP). Seeds were considered germinated when the healthy, white radicle had emerged through the integument and reached more than 1 mm in length (Bewley and Black, 1978). The germination rate index (GRI) was also calculated for each treatment using the following equation: (GRI) ¼ (G1/1)+(G2/2)+ y +(Gx/x), where G is the germination on each day after placement, and 1,2, y, x represents the corresponding day of germination (Esechie, 1994). Corrected germination rate index (CGRI) was obtained by dividing GRI by the final germination percentage or FGP (GP at 16 days after the beginning of seed incubation for our experiments) and multiplying by 100. The number of days lapsed to reach 50% of the FGP (GT50), another widely used index in order to compare relative rate of germination, was also calculated (Hsu et al., 1985). Subsequently, two pot experiments were conducted in a growth chamber. Day/night length, air temperature and relative humidity were: 14/10 h, 28/18 1C and 50/70%, respectively, and a PPFD of about 320 mmol m2 s1. Fifteen pre-germinated seeds of each treatment reaching a radicle length of 2–4 cm were sown at 4 cm depth. One seed was planted in each plastic pot (15 cm in diameter), filled with 2.4 l mixture of peat and perlite (2:1, v/v). Since marama seeds (even pre-germinated) seem to require a large oxygen supply to prevent rotting. Watering was carried out with 200 ml of distilled water in each pot every 4 days. The number of days from sowing to emergence was recorded for all the seedlings. The percentages of germination and emergence (after angular transformation) and the other data were subjected to ANOVA using the Statgraphics statistical software package (v.5.0, Statistical Graphics Corporation, Englewood Cliffs, NJ, USA). Mean comparison was performed using Fisher’s least-significant difference (LSD) method (po0.05). 3. Results The effects of the several treatments on seed germination progress for the first and second experiment are given in Tables 1 and 2, respectively. Immersion in water for 20 h or in sulphuric acid for 20 min increased significantly (po0.05) the FGP of T. esculentum seeds from 63% (control) to 86% and 75%, respectively. The rest treatments resulted to intermediate values of germination (slightly higher than controls). Furthermore, seed immersion in water for 10 h and in sulphuric acid for 20 min successively resulted to the lowest percentages of germination during the whole experimental period (Table 1). The second germination experiment revealed that the mechanical scarification was the most effective treatment to promote germination (po0.05) and a GP of 100% was already achieved after an incubation of 12 days, while in the same time the corresponding percentages for all the other seeds ranged between 40% and 63%. The FGP after an immersion in sulphuric acid for 20 min was also high (Table 2). The germination rate of mechanically scarified seeds was greatly enhanced (highest CGRI and 50% germination after 2 days; po0.05, Table 3). Additionally, immersion in water for 20 h, in sulphuric acid for 20 min and in water for 10 h followed by gibberellic
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Table 1 Time course for the germination of T. esculentum seeds in response to different pretreatments Days after seed placement
2 4 6 8 10 12 14 16a
Pretreatment Control
Water for 20 h
Sulphuric acid for 20 min
Water for 10 h and sulphuric acid for 20 min
Gibberellic acid for 40 min
Water for 10 h and gibberrellic acid for 40 min
15 25 33 38 38 39 50 63
28 42 49 61 63 78 81 86
15 36 52 63 63 69 69 75
5d 11 c 24 c 28 c 38 c 40 c 40 c 48 d
10 27 32 42 50 63 63 64
18 32 37 56 63 69 69 73
bc b b bc c c c c
a a a a a a a a
bc a a a a ab b b
cd b bc b b b b c
b ab b a a ab b bc
Germination percentages (%) are means of eight replications (duration of the first germination experiment ¼ 16 days). Different case letters in the same line indicate significant differences (po0.05, Fisher’s LSD test) between pretreatments. All per cent germination data of the first experiment were angular transformed for analysis. a Seed germination at 16 days after seed placement represents the final germination percentage (FGP).
Table 2 Time course for the germination of T. esculentum seeds in response to different pretreatments Days after seed placement
2 4 6 8 10 12 14 16a
Pretreatment Control
Sulphuric acid for 20 min
Sulphuric acid for 40 min
Gibberellic acid for 40 min
Mechanical scarification
19 25 34 36 40 40 46 59
25 35 44 51 61 63 68 73
20 30 40 45 55 58 61 64
21 29 35 43 50 58 59 63
53 a 69 a 89 a 93 a 95 a 100 a 100 a 100 a
b b b b c c c c
b b b b b b b b
b b b b bc b bc bc
b b b b bc b bc bc
Germination percentages (%) are means of eight replications (duration of the second germination experiment ¼ 16 days). Different case letters in the same line indicate significant differences (po0.05, Fisher’s LSD test) between pretreatments. All per cent germination data of the second experiment were angular transformed for analysis. a Seed germination at 16 days after seed placement represents the final germination percentage (FGP).
acid solution for 40 min were also effective. Immersion in water followed by acid scarification of the seeds clearly resulted in a low rate of germination (the CGRI value was lower than the corresponding value for the control batch). Besides, in Table 3 it is shown that although the emergence percentage and rate of untreated marama seeds (control) were relatively satisfactory, the mechanical scarification method (followed by the immersion in water for 20 h and in sulphuric acid for 20 min) resulted to the optimization of marama emergence.
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Table 3 Corrected germination rate index (CGRI), number of days to 50% of the final germination (GT50) of T. esculentum seeds, mean emergence percentage and days from sowing to emergence as affected by seed pretreatment (A: first and B: second germination and emergence assay) Pretreatments
A Control Water for 20 h Sulphuric acid for 20 min Water for 10 h and sulphuric acid for 20 min Gibberellic acid for 40 min Water for 10 h and gibberrellic acid for 40 min B Control Sulphuric acid for 20 min Sulphuric acid for 40 min Gibberellic acid for 40 min Mechanical scarification
CGRI (% day1)
GT50 (days)
Mean emergence (%)
Average days of emergence
61.21 74.71 72.95 53.63
6a 5.5 bc 4.5 d 5.75 ab
73.33 b 80 a 80 a 73.33 b
10 a 9b 8c 10 a
64.19 b 70.84 a
5.25 c 5.5 bc
66.67 c 66.67 c
9b 9b
68.17 b 76.34 b 75.75 b 74.51 b 101.43 a
5.75 a 5.5 a 4.75 a 5.5 a 2.25 b
73.33 bc 80 ab 66.67 c 66.67 c 86.67 a
10 a 8c 9b 9b 7d
b a a c
All data are means of all replications (duration of each germination and emergence experiment ¼ 16 days). For each experiment different case letters in the same column indicate significant differences (po0.05, Fisher’s LSD test) between pretreatments.
4. Discussion and conclusions The hard seed coat of many leguminous species offers important ecological advantages. This feature favours the accumulation of persistent seed banks in the soil, spreads germination over time and increases the chance that some seeds will germinate, establish and complete the life cycle successfully (Gutterman, 1993). However, high, rapid and uniform seed germination is pre-adequate for the successful establishment of the underutilized plant species such as T. esculentum. In the present study, abrasion of the seed with sandpaper increased significantly the germination and emergence percentage and was the most rapid scarification treatment. The beneficial effect of mechanical scarification on seed germination is common at many hard-coated perennial legumes widespread in arid and semi-arid zones (Demel, 1998; Sy et al., 2001; Vilela and Ravetta, 2001; Ortega Baes et al., 2002). Similar trends of another method of mechanical scarification (cracking of marama seeds) have been reported in the study of Lebutswe et al. (2003). In contrast to the abrasion with sandpaper, cracking gave a low emergence rate (52%), although the germination of the cracked seeds was very high (90–100%). It is plausible to ascribe this difference to a possible damage of the seed by cracking, which resulted to an insufficient emergence of the germinated seeds. It seems that this damage can be possibly avoided by the use of sandpaper (gentler method of mechanical scarification). In a contrary way to some—and in accordance to—other hard-seeded legumes native to arid and semi-arid environments, water immersion was very effective on marama seed
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germination and emergence (Roussel, 1996; Sy et al., 2001). This finding is in agreement with the results of the study of Lebutswe et al. (2003) on marama. Concerning the germination promotion of the seeds treated with sulphuric acid and their possible damages caused by prolonged immersion or low integumental resistance are common in marama (Lebutswe et al., 2003) and other arid-adapted hardcoated legumes (Demel, 1998; Sy et al., 2001; Vilela and Ravetta, 2001; Ortega Baes et al., 2002). Gibberellic acid treatment after water immersion resulted to higher GP than single gibberellic acid immersion. Therefore, it seems likely that the primary control of germination in marama seeds resides in the seed coats, and that GA3 action is promoted by a previous seed coat disruption, likewise Capparis spinosa L. (Sozzi and Chiesa, 1995). Nevertheless, the use of gibberellic acid was significantly less effective on marama germination and emergence than the other applied treatments. In addition, there was a significant positive correlation between GP (FGP) and germination rate (CGRI) (r2 ¼ 0.97, po0.005), suggesting that the rapid germination was associated with the high GP. The CGRI was also significantly correlated with GT50 (r2 ¼ 0.97, po0.01) indicating that CGRI was a good index in order to express relative speed of germination. On the contrary, marama germination was not closely associated with the emergence of the seedlings. The results from the present study are in agreement with previous reports, providing evidence that marama seeds have little seed coat dormancy and do not involve physiological dormancy (Lebutswe et al., 2003), as long as they do not require a dormance-breaking pre-treatment per se in order to germinate (Baskin and Baskin, 2004). The germination rate and percentage of T. esculentum can be greatly increased by subjecting the seeds to mechanical scarification, water immersion (Lebutswe et al., 2003) or acid treatment, such as in several legumes of arid regions (Demel, 1998). Actually, the partial removal of the seed covering structures by means of the massive abrasion can be considered as the most effective treatment in stimulating germination and emergence. Furthermore, mechanical scarification not only greatly enhanced germination, but also increased significantly the germination rate, resulting to a rapid and high emergence of vigorous seedlings and consequent successful establishment. The scarification of marama seeds with sandpaper before planting could be used in the field to improve stand establishment, since this method not only resulted to high germination and emergence percentage, but also it can be rapidly and massively applied. The low seed set of this species (Hartley et al., 2002) combined with the high risk of its over-exploitation and its potential establishment as a drought tolerant crop, confirm the significant role of similar studies. Field experiments and further laboratory studies which are already being conducted, must be continued in order to validate our results and optimize marama seed germination and seedling emergence, growth and establishment.
Acknowledgements This work was carried out as part of the MARAMA project funded by the European Commission; contract number ICA4-CT-2000-30010. The authors wish to thank Dr. G.M. Ramolemana and Botswana College of Agriculture for kindly providing T. esculentum seeds.
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References Baskin, J.M., Baskin, C.C., 2004. A classification system for seed dormancy. Seed Science Research 14, 1–16. Bewley, J.D., Black, M., 1978. Physiology and Biochemistry of Seeds, vol. 1. Springer, Berlin, Heidelberg, New York, 306pp. Bousquet, J., 1981-1982. The morama bean of the Kalahari Desert as a potential food crop, with a summary of current research in Texas. Desert Plants 3, 213–215. Bower, N., Hertel, K., Oh, J., Storey, R., 1988. Nutritional evaluation of marama bean (Tylosema esculentum, Fabaceae): analysis of the seed. Economic Botany 42 (4), 533–540. Cavanagh, T., 1987. Germination of hard-seeded species (Order Fabales). In: Langkamp, P. (Ed.), Germination of Australian Native Plant Seed. AMIRA, Melbourne/Sydney, pp. 58–70. Demel, T., 1998. Germination of Acacia origena, A. pilispina and Pterobium stellatum in response to different presowing seed treatments, temperature and light. Journal of Arid Environments 38, 551–560. Esechie, H., 1994. Interaction of salinity and temperature on the germination of sorghum. Journal of Agronomy and Crop Science 172, 194–199. Gutterman, Y., 1993. Seed Germination in Desert Plants. Berlin, Springer, 253pp. Hartley, M.L., Tshamekeng, E., Thomas, S.M., 2002. Functional heterostyly in Tylosema esculentum (Caesalpinioidae). Annals of Botany 89, 67–76. Hsu, F.H., Nelson, C.J., Matches, A.G., 1985. Temperature effects on germination of perennial warm-season forage grasses. Crop Science 25, 215–220. Hyde, E., 1954. The function of the hilum in some Papilionaceae in relation to the ripening of the seed and the permeability of the testa. Annals of Botany (London) 18, 241–256. Keegan, A.B., Vanstaden, J., 1981. Marama Bean, Tylosema Esculentum, a plant worthy of cultivation. South African Journal of Science 77 (9), 387. Ketshajwang, K.K., Holmback, J., Yeboah, S.O., 1998. Quality and compositional studies of some edible Leguminosae seed oils in Botswana. Journal of the American Oil Chemist’s Society 75, 741–743. Lebutswe, T., Ramolemana, G.M., Machacha, S., Tsopito, C.M., 2003. Effects of different dormancy-breaking treatments on the germination and emergence of morama bean (Tylosema esculentum Burch.), Botswana. UNISWA Journal of Agriculture. National Academy of Sciences. National Research Council, 1979. p. 68–74 in Tropical Legumes: resources for the future. Report of the Ad Hoc Panel of the Advisory Committee on Technology Innovation Board on Science and Technology for International Development, Commission on International Relations, National Academy of Sciences, Washington, DC. Ortega Baes, P., de Viana, M.L., Suhring, S., 2002. Germination in Prosopis ferox seeds: effects of mechanical, chemical and biological scarificators. Journal of Arid Environments 50, 185–189. Roussel, J., 1996. Pe´pinie`re et plantations forestie`res en Afrique tropicale se`che. ISRA/CIRAD, Dakar, Senegal, 435pp. Sozzi, G.O., Chiesa, A., 1995. Improvement of caper (Capparis spinosa L.) seed germination by breaking seed coat-induced dormancy. Scientia Horticulturae 62, 255–261. Sy, A., Grouzis, M., Danthu, P., 2001. Seed germination of seven Sahelian legume species. Journal of Arid Environments 49, 875–882. Vilela, A.E., Ravetta, D.A., 2001. The effect of seed scarification and soil-media on germination, growth, storage, and survival of seedlings of five species of Prosopis L. (Mimosaceae). Journal of Arid Environments 48, 171–184.
Journal of Arid Environments 68 (2007) 501–507 www.elsevier.com/locate/jnlabr/yjare
Short communication
Germination and emergence of the hard seed coated Tylosema esculentum (Burch) A. Schreib in response to different pre-sowing seed treatments I.S. Travlos, G. Economou, A.I. Karamanos Agricultural University of Athens, Department of Crop Production, Laboratory of Agronomy, 75, Iera Odos st., 11855 Athens, Greece Received 17 November 2005; received in revised form 18 May 2006; accepted 3 July 2006 Available online 15 August 2006
Abstract Marama bean (Tylosema esculentum (Burch) L. Schreib) is an underutilized drought-tolerant legume native to the arid and semi-arid regions of southern Africa, which produces protein- and oilrich seed and tubers with potential for use as human food and animal fodder. Laboratory experiments were conducted in Athens, in order to investigate germination behaviour of untreated seeds and seeds subjected to different dormancy-breaking treatments and subsequent emergence of the seedlings. The effect of some scarification methods on seed germination was estimated at 30 1C. Most treatments, likewise control, resulted to relatively high germination and emergence percentages, confirming that marama seeds have no physiological dormancy. The speed and percentage of T. esculentum seed germination was greatly increased by mechanical massive scarification of the seeds with sandpaper. These highly positive responses of marama seeds to the mechanical treatment clearly indicate that there is a moderate coat-imposed dormancy in this species. Immersion in water for 20 h and in concentrated sulphuric acid for 20 min were also some of the most effective treatments. r 2006 Elsevier Ltd. All rights reserved. Keywords: Seed dormancy; Hard seed coat; Mechanical scarification; Marama; Africa
Corresponding author. Tel./fax:+3 210 529 4482.
E-mail addresses: [email protected] (I.S. Travlos), [email protected] (G. Economou), [email protected] (A.I. Karamanos). 0140-1963/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2006.07.001
ARTICLE IN PRESS 502
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1. Introduction Marama bean (Tylosema esculentum (Burch.) A. Schreib) is a wild perennial legume, indigenous to the arid and semi-arid grasslands of southern Africa (Bousquet, 1981-1982). The remarkable socio-economic value of the plant in African communities is mainly explained by the fact that the bean is highly nutritious when cooked (Keegan and Vanstaden, 1981), with protein and oil content comparable to soybean (Glycine max) and groundnut (Arachis hypogaea), respectively (Bower et al., 1988; Ketshajwang et al., 1998). Because of its great ability to survive under unfavourable conditions it can be considered suitable for cultivation, especially under preventive conditions for other crops (National Academy of Sciences, 1979; Keegan and Vanstaden, 1981). Marama seeds are relatively large, with a hard seed coat, able to resist mechanical damage, likewise seeds of other perennial legume species (Hyde, 1954). In the nature, there are various biotic and abiotic factors that promote seed scarification, such as mechanical abrasion of the seed coat by sand and rocks in water courses (Vilela and Ravetta, 2001). Under laboratory conditions and in agriculture, other methods to render permeable hard seed coats have been adopted and discussed in detail by Cavanagh (1987). The objective of this study was to assess the influence of several seed scarification methods on germination and emergence of T. esculentum. The potential establishment of this species as a crop and the risk of its over-exploitation, with the attendant loss of biodiversity (seed is gathered from the wild and widely consumed by local people and range animals) make imperative the need of such a study, in order to optimize its seed germination and first growth.
2. Materials and methods T. esculentum seeds were collected directly from the wild in Letlhakane, Botswana (211 340 S; 251 420 E) and stored at 4 1C and 50% RH until their use 10 months later (fresh weight was ranged between 2 and 3 g). Two germination experiments were carried out in completely randomized designs in incubators (Conviron T 38/Lb/AP) at constant temperature (30 1C) and total darkness. In the first experiment, the effects of the following treatments on seed germination were evaluated: (1) immersion in water at room temperature (25–30 1C) for 20 h; (2) immersion in concentrated (95%) sulphuric acid for 20 min followed by thorough rinsing with running water; (3) immersion in water for 10 h followed by immersion in concentrated sulphuric acid for 20 min and thorough rinsing with running water; (4) immersion in a gibberellic acid (GA3) solution (100 mg GA3 l1 water) for 40 min and (5) immersion in water for 10 h and in the above-mentioned GA3 aqueous solution for 40 min successively. In the second germination assay, seeds were subjected to (1) immersion in concentrated (95%) sulphuric acid for 20 min followed by thorough rinsing with running water; (2) immersion in concentrated (95%) sulphuric acid for 40 min followed by thorough rinsing with running water; (3) immersion in a gibberellic acid (GA3) solution (100 mg GA3 l1 water) for 40 min and (4) mechanical scarification (massive abrasion of the seeds between two sheets of sandpaper). Eight replicates (Petri dishes) were used for each treatment. Five seeds were placed between two Whatman no. 1 paper filter disks (Whatman Ltd., Maidstone, England) in
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each 9-cm Petri dish, and 5 ml of distilled water was added whenever there was a need to keep filter papers moist. Seed germination was recorded every other day and expressed as a percentage of the total number of tested seeds (germination percentage, GP). Seeds were considered germinated when the healthy, white radicle had emerged through the integument and reached more than 1 mm in length (Bewley and Black, 1978). The germination rate index (GRI) was also calculated for each treatment using the following equation: (GRI) ¼ (G1/1)+(G2/2)+ y +(Gx/x), where G is the germination on each day after placement, and 1,2, y, x represents the corresponding day of germination (Esechie, 1994). Corrected germination rate index (CGRI) was obtained by dividing GRI by the final germination percentage or FGP (GP at 16 days after the beginning of seed incubation for our experiments) and multiplying by 100. The number of days lapsed to reach 50% of the FGP (GT50), another widely used index in order to compare relative rate of germination, was also calculated (Hsu et al., 1985). Subsequently, two pot experiments were conducted in a growth chamber. Day/night length, air temperature and relative humidity were: 14/10 h, 28/18 1C and 50/70%, respectively, and a PPFD of about 320 mmol m2 s1. Fifteen pre-germinated seeds of each treatment reaching a radicle length of 2–4 cm were sown at 4 cm depth. One seed was planted in each plastic pot (15 cm in diameter), filled with 2.4 l mixture of peat and perlite (2:1, v/v). Since marama seeds (even pre-germinated) seem to require a large oxygen supply to prevent rotting. Watering was carried out with 200 ml of distilled water in each pot every 4 days. The number of days from sowing to emergence was recorded for all the seedlings. The percentages of germination and emergence (after angular transformation) and the other data were subjected to ANOVA using the Statgraphics statistical software package (v.5.0, Statistical Graphics Corporation, Englewood Cliffs, NJ, USA). Mean comparison was performed using Fisher’s least-significant difference (LSD) method (po0.05). 3. Results The effects of the several treatments on seed germination progress for the first and second experiment are given in Tables 1 and 2, respectively. Immersion in water for 20 h or in sulphuric acid for 20 min increased significantly (po0.05) the FGP of T. esculentum seeds from 63% (control) to 86% and 75%, respectively. The rest treatments resulted to intermediate values of germination (slightly higher than controls). Furthermore, seed immersion in water for 10 h and in sulphuric acid for 20 min successively resulted to the lowest percentages of germination during the whole experimental period (Table 1). The second germination experiment revealed that the mechanical scarification was the most effective treatment to promote germination (po0.05) and a GP of 100% was already achieved after an incubation of 12 days, while in the same time the corresponding percentages for all the other seeds ranged between 40% and 63%. The FGP after an immersion in sulphuric acid for 20 min was also high (Table 2). The germination rate of mechanically scarified seeds was greatly enhanced (highest CGRI and 50% germination after 2 days; po0.05, Table 3). Additionally, immersion in water for 20 h, in sulphuric acid for 20 min and in water for 10 h followed by gibberellic
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Table 1 Time course for the germination of T. esculentum seeds in response to different pretreatments Days after seed placement
2 4 6 8 10 12 14 16a
Pretreatment Control
Water for 20 h
Sulphuric acid for 20 min
Water for 10 h and sulphuric acid for 20 min
Gibberellic acid for 40 min
Water for 10 h and gibberrellic acid for 40 min
15 25 33 38 38 39 50 63
28 42 49 61 63 78 81 86
15 36 52 63 63 69 69 75
5d 11 c 24 c 28 c 38 c 40 c 40 c 48 d
10 27 32 42 50 63 63 64
18 32 37 56 63 69 69 73
bc b b bc c c c c
a a a a a a a a
bc a a a a ab b b
cd b bc b b b b c
b ab b a a ab b bc
Germination percentages (%) are means of eight replications (duration of the first germination experiment ¼ 16 days). Different case letters in the same line indicate significant differences (po0.05, Fisher’s LSD test) between pretreatments. All per cent germination data of the first experiment were angular transformed for analysis. a Seed germination at 16 days after seed placement represents the final germination percentage (FGP).
Table 2 Time course for the germination of T. esculentum seeds in response to different pretreatments Days after seed placement
2 4 6 8 10 12 14 16a
Pretreatment Control
Sulphuric acid for 20 min
Sulphuric acid for 40 min
Gibberellic acid for 40 min
Mechanical scarification
19 25 34 36 40 40 46 59
25 35 44 51 61 63 68 73
20 30 40 45 55 58 61 64
21 29 35 43 50 58 59 63
53 a 69 a 89 a 93 a 95 a 100 a 100 a 100 a
b b b b c c c c
b b b b b b b b
b b b b bc b bc bc
b b b b bc b bc bc
Germination percentages (%) are means of eight replications (duration of the second germination experiment ¼ 16 days). Different case letters in the same line indicate significant differences (po0.05, Fisher’s LSD test) between pretreatments. All per cent germination data of the second experiment were angular transformed for analysis. a Seed germination at 16 days after seed placement represents the final germination percentage (FGP).
acid solution for 40 min were also effective. Immersion in water followed by acid scarification of the seeds clearly resulted in a low rate of germination (the CGRI value was lower than the corresponding value for the control batch). Besides, in Table 3 it is shown that although the emergence percentage and rate of untreated marama seeds (control) were relatively satisfactory, the mechanical scarification method (followed by the immersion in water for 20 h and in sulphuric acid for 20 min) resulted to the optimization of marama emergence.
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Table 3 Corrected germination rate index (CGRI), number of days to 50% of the final germination (GT50) of T. esculentum seeds, mean emergence percentage and days from sowing to emergence as affected by seed pretreatment (A: first and B: second germination and emergence assay) Pretreatments
A Control Water for 20 h Sulphuric acid for 20 min Water for 10 h and sulphuric acid for 20 min Gibberellic acid for 40 min Water for 10 h and gibberrellic acid for 40 min B Control Sulphuric acid for 20 min Sulphuric acid for 40 min Gibberellic acid for 40 min Mechanical scarification
CGRI (% day1)
GT50 (days)
Mean emergence (%)
Average days of emergence
61.21 74.71 72.95 53.63
6a 5.5 bc 4.5 d 5.75 ab
73.33 b 80 a 80 a 73.33 b
10 a 9b 8c 10 a
64.19 b 70.84 a
5.25 c 5.5 bc
66.67 c 66.67 c
9b 9b
68.17 b 76.34 b 75.75 b 74.51 b 101.43 a
5.75 a 5.5 a 4.75 a 5.5 a 2.25 b
73.33 bc 80 ab 66.67 c 66.67 c 86.67 a
10 a 8c 9b 9b 7d
b a a c
All data are means of all replications (duration of each germination and emergence experiment ¼ 16 days). For each experiment different case letters in the same column indicate significant differences (po0.05, Fisher’s LSD test) between pretreatments.
4. Discussion and conclusions The hard seed coat of many leguminous species offers important ecological advantages. This feature favours the accumulation of persistent seed banks in the soil, spreads germination over time and increases the chance that some seeds will germinate, establish and complete the life cycle successfully (Gutterman, 1993). However, high, rapid and uniform seed germination is pre-adequate for the successful establishment of the underutilized plant species such as T. esculentum. In the present study, abrasion of the seed with sandpaper increased significantly the germination and emergence percentage and was the most rapid scarification treatment. The beneficial effect of mechanical scarification on seed germination is common at many hard-coated perennial legumes widespread in arid and semi-arid zones (Demel, 1998; Sy et al., 2001; Vilela and Ravetta, 2001; Ortega Baes et al., 2002). Similar trends of another method of mechanical scarification (cracking of marama seeds) have been reported in the study of Lebutswe et al. (2003). In contrast to the abrasion with sandpaper, cracking gave a low emergence rate (52%), although the germination of the cracked seeds was very high (90–100%). It is plausible to ascribe this difference to a possible damage of the seed by cracking, which resulted to an insufficient emergence of the germinated seeds. It seems that this damage can be possibly avoided by the use of sandpaper (gentler method of mechanical scarification). In a contrary way to some—and in accordance to—other hard-seeded legumes native to arid and semi-arid environments, water immersion was very effective on marama seed
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germination and emergence (Roussel, 1996; Sy et al., 2001). This finding is in agreement with the results of the study of Lebutswe et al. (2003) on marama. Concerning the germination promotion of the seeds treated with sulphuric acid and their possible damages caused by prolonged immersion or low integumental resistance are common in marama (Lebutswe et al., 2003) and other arid-adapted hardcoated legumes (Demel, 1998; Sy et al., 2001; Vilela and Ravetta, 2001; Ortega Baes et al., 2002). Gibberellic acid treatment after water immersion resulted to higher GP than single gibberellic acid immersion. Therefore, it seems likely that the primary control of germination in marama seeds resides in the seed coats, and that GA3 action is promoted by a previous seed coat disruption, likewise Capparis spinosa L. (Sozzi and Chiesa, 1995). Nevertheless, the use of gibberellic acid was significantly less effective on marama germination and emergence than the other applied treatments. In addition, there was a significant positive correlation between GP (FGP) and germination rate (CGRI) (r2 ¼ 0.97, po0.005), suggesting that the rapid germination was associated with the high GP. The CGRI was also significantly correlated with GT50 (r2 ¼ 0.97, po0.01) indicating that CGRI was a good index in order to express relative speed of germination. On the contrary, marama germination was not closely associated with the emergence of the seedlings. The results from the present study are in agreement with previous reports, providing evidence that marama seeds have little seed coat dormancy and do not involve physiological dormancy (Lebutswe et al., 2003), as long as they do not require a dormance-breaking pre-treatment per se in order to germinate (Baskin and Baskin, 2004). The germination rate and percentage of T. esculentum can be greatly increased by subjecting the seeds to mechanical scarification, water immersion (Lebutswe et al., 2003) or acid treatment, such as in several legumes of arid regions (Demel, 1998). Actually, the partial removal of the seed covering structures by means of the massive abrasion can be considered as the most effective treatment in stimulating germination and emergence. Furthermore, mechanical scarification not only greatly enhanced germination, but also increased significantly the germination rate, resulting to a rapid and high emergence of vigorous seedlings and consequent successful establishment. The scarification of marama seeds with sandpaper before planting could be used in the field to improve stand establishment, since this method not only resulted to high germination and emergence percentage, but also it can be rapidly and massively applied. The low seed set of this species (Hartley et al., 2002) combined with the high risk of its over-exploitation and its potential establishment as a drought tolerant crop, confirm the significant role of similar studies. Field experiments and further laboratory studies which are already being conducted, must be continued in order to validate our results and optimize marama seed germination and seedling emergence, growth and establishment.
Acknowledgements This work was carried out as part of the MARAMA project funded by the European Commission; contract number ICA4-CT-2000-30010. The authors wish to thank Dr. G.M. Ramolemana and Botswana College of Agriculture for kindly providing T. esculentum seeds.
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