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Seed germination of Lasia spinosa as a function of temperature, light, desiccation, and storage
Aquatic Botany 89 (2008) 352–356
Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Seed germination of Lasia spinosa as a function of temperature, light, desiccation, and storage An-jun Tang a,b, Chun-lin Long a,* a b
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China Graduate School of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 April 2007 Received in revised form 7 March 2008 Accepted 3 April 2008 Available online 12 April 2008
Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 8C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 8C, after 60 days at 10 8C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 8C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H2O g1 dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H2O g1 dry wt moisture content. Seeds stored at 0.58 g H2O g1 dry wt moisture content at four constant temperatures (4, 10, 15, and 18 8C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H2O g1 dry wt moisture content could be effectively stored for a few months between 10 and 15 8C although the most appropriate temperature for wet storage appears to be 10 8C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 8C. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Lasia spinosa Desiccation tolerance Recalcitrant seeds Seed germination Seed storage Tropical swamp
1. Introduction At present, some tropical wetland species in China are at risk because of deforestation and environmentally unsustainable land use (Tan and Ni, 2006). For example, Lasia spinosa (L.) Thwait. (Araceae), a valuable swamp species which can be used as an ornamental, medicine and vegetable, is decreasing greatly due to excessive extraction and degradation of its habitat (Li, 1979). One method of preserving genetic diversity of this species is to store its seeds in the seed-gene bank. However, before seed storage can be implemented successfully, a full understanding of the requirements for seed germination and desiccation tolerance is essential. Seed sensitivity to desiccation is more common among species growing in permanently moist areas, such as tropical rainforest (Tweddle et al., 2003). In addition, some but not all wetland species produce recalcitrant (desiccation sensitive) seeds (Pammenter and Berjak, 2000), including many riparian species, such as Ravenea
* Corresponding author. Fax: +86 871 5223233. E-mail address: [email protected] (C.-l. Long). 0304-3770/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2008.04.003
rivularis (Arecaceae) (Rakondranony et al., 2006). Finally, some understorey herbs also produce desiccation intolerant seeds (Suzuki et al., 2007). Recalcitrant seeds tend to be produced by trees, are relatively large in size and shed during the rainy season (Pritchard et al., 2004) and germinate relatively rapidly, unhindered by the presence of a relatively thin seed coat (Pritchard et al., 2004; Daws et al., 2005, 2006). Such adaptations increase the likelihood of ensuring continuous moisture, thus avoiding seed desiccation stress (Mata and Moreno-Casasola, 2005). As demonstrated by many studies, seasonal variations in temperature regulate the timing of germination in some aquatic species (Welling et al., 1988; Baskin et al., 1996; Brenchley and Probert, 1996, 1998; Mata and Moreno-Casasola, 2005). Additionally, light significantly affects seed germination of some plants (Pons, 2000). To facilitate successful restoration of swampy ecosystems, information on seed dormancy and germination requirements and storability of seeds of critical species is needed. Thus, we investigated germination characteristics and desiccation sensitivity of L. spinosa seeds. Since L. spinosa is an aquatic species that disperses its seeds into relatively open very moist environments, we hypothesized that seeds may require light for germination and that they may be desiccation intolerant.
A.-j. Tang, C.-l. Long / Aquatic Botany 89 (2008) 352–356
353
2. Materials and methods
2.5. Effect of storage on germination
2.1. Study species and area
Seeds of L. spinosa with a moisture content of 0.58 g H2O g1 dry wt and high viability were stored hermetically at 4, 10, 15, and 18 8C for up to 6 months. After 1, 3 and 6 months, 100 seeds were removed from storage and tested for germination in light at 25 8C, using four replicates of 25 seeds each.
L. spinosa is a perennial evergreen herb growing on wet, nutrientrich soils near or in ditches, brooks, ponds or grass-clusters, commonly characterized by seasonally changing water levels. Such sites are common near villages or on moist farmland in southeastern Asian, including China, Thailand, and Malaya (Li, 1979). Plants of L. spinosa produce one-seeded berries that mature in February. The study was carried out in the Xishuangbanna Tropical Botanical Garden (XTBG) (218410 N, 1018250 E, 570 m altitude) of the Chinese Academy of Sciences. XTBG is located in southern Yunnan province, where annual rainfall is about 1493 mm with a dry season from November to next April. Mean annual temperature is 21.8 8C, and the hottest and coldest months are June and January, respectively, with a mean temperature of 25.7 and 16 8C, respectively (http://cbis.xtbg.ac.cn/db/PDF_LYR/476265.pdf). 2.2. Seed collection and storage To obtain a sufficient quantity of seeds, fruits were collected from 100 plants growing in the XTBG on 6 February 2004. Within 1 day after collection, seeds were separated carefully from the berries by hand, cleaned and stored in paper bags at room temperature (20 2 8C) for 1 or 2 days, except for those used to determine initial moisture content. Also, we measured the dimension (length breadth) of 100 seeds and the 1000-seed weight. 2.3. Germination requirements
2.6. Data analysis Percentage germination was calculated for all tests. Analysis of variance was used to determine the effects of factors on germination percentage (arcsine transformed for linearity). Differences between means were determined using least significant difference (LSD), or standard errors of the mean values. 3. Results 3.1. Germination requirements Optimal temperature for germination (97%) was 25 8C, but seeds also germinated to 80% at 30 8C (Fig. 1). Daily fluctuation of 20/30 8C was also effective for a high germination response (94% germination) (Fig. 1). Below 20 8C, percentage germination was very low, and no germination occurred at 10 8C even after 60 days of incubation. When seeds were transferred from 10 to 25 8C, they germinated to 17 3.2%, 29 4.7% and 78 2.8% after 5, 15 and 20 days, respectively. Freshly harvested seeds germinated to 97% and 86% at 25 8C in light and in dark, respectively (Fig. 2). Although final
Four replicates of 25 freshly collected seeds each were placed in 9 cm diameter glass Petri dishes on filter paper moistened with distilled water and placed incubators equipped with a cool, white fluorescent light (HPG-280B, Har’erbin, China) providing a photosynthetic photon flux density (PPFD) measured with LI-1400 Data logger (LI-COR, USA) of approximately 44 mmol m2 s1 over the waveband 400–700 nm. Seeds were exposed to a daily 14 h daily photoperiod at constant temperatures of 10, 15, 20, 25, 30, 35, and a daily 14 h fluctuating cycle of 20/30 8C. Germination counts were made daily for 30–80 days depending on germination rate in relation to temperature. At the end of a 60-day incubation period at 10 8C, seeds that had not germinated were transferred to 25 8C for 20 days. In addition, we compared germination under light and continuous dark at 25 8C using a green safe light to examine seeds incubated in darkness. Distilled water was added as required to ensure that moisture was non-limiting for germination. Seeds were recorded as germinated when the protruding radicle was 2 mm in length. 2.4. Desiccation sensitivity Moisture content for freshly collected seeds was calculated immediately after cleaning according to recommended protocols of the International Seed Testing Association (ISTA, 1999). During slow dehydration, seeds were spread in a monolayer and dried for up to 3 weeks in a dry room operating at 15 8C and 45% relatively humidity (RH). Moisture contents of all seed lots were determined gravimetrically before and after drying in a 103 8C oven for 17 h (ISTA, 1999). At each sample time during the drying curve, we withdrew 15 seeds to determine seed moisture content. Seed moisture content was expressed on the dry weight basis (i.e., g H2O g1 dry wt). Each time seed moisture was determined, seeds were tested for germination in light at 25 8C, using four replicates of 25 seeds each.
Fig. 1. Effects of temperature on germination of Lasia spinosa seeds. Bars indicate standard deviations.
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A.-j. Tang, C.-l. Long / Aquatic Botany 89 (2008) 352–356 Table 1 Effects of storage temperature and duration on viability of Lasia spinosa seeds with 0.58 g H2O g1 dry wt moisture content Storage period (months)
Germination (%) (S.D.) after storage 4 8C
10 8C
15 8C
18 8C
1 3 6
83 0.58a 37 2.83b 18 1.42c
86 2.2a 76 0.8a 74 1.5a
84 3.8a 81 0.7a 77 1.3a
0 0 0
Germination percentage is the mean value of four replicates 1 S.D. For each storage period, differences between temperatures (P < 0.05) are indicated by different lowercase letters. In each column values followed by the same letter are not significantly different (P < 0.05).
Fig. 2. Effect of light (14 h daily photoperiod) and continuous darkness on germination of Lasia spinosa seeds at 25 8C.
declined gradually, with an increase in storage duration. The greatest decrease in viability (from 83% to 18%) occurred at 4 8C (Table 1). Seed viability was not significantly reduced during storage at 10 or 15 8C, suggesting that prolonging storage duration would not impair seed viability at 10 or 15 8C. 4. Discussion
germination percentages in the light and dark did not significantly differ (P > 0.05), dark germination took more than twice as long as that in light (Fig. 2). 3.2. Desiccation sensitivity L. spinosa seeds were relatively large and heavy, with a mean dimension of 100 seeds of 1.1 cm 0.8 cm (length breadth) and a 1000-seed weight of 306.4 2.2 g. Water content of L. spinosa seeds sharply decreased with drying time at 15 8C and 45% RH (Fig. 3). As seed moisture content decreased from 2.02 to 0.58 g H2O g1 dry wt, seed germination percentage remained high (Fig. 3). However, further drying to 0.13 g H2O g1 dry wt caused almost complete loss of viability. The critical moisture for viability loss, i.e., the mid-point, was about 0.35 g H2O g1 dry wt (or 26% moisture content, fresh weight basis). 3.3. Effect of storage on germination Storage duration combined with temperature had strong effects on the germination of L. spinosa seeds with 0.58 g H2O g1 dry wt moisture content (Table 1). No germination occurred after 1, 3 or 6 months of storage at 18 8C. At 4, 10 and 15 8C, seed viability
Fig. 3. Relationship between moisture content of Lasia spinosa seeds and drying time at 15 8C and 45% RH (*) and effects of moisture content on germination of L. spinosa seeds at 25 8C (*). Bars indicate standard deviations.
Freshly harvested mature seeds of L. spinosa seeds were nondormant and germinated to high percentages at 25 and 30 8C. When seeds were transferred from 10 to 25 8C after 60 days, germination increased from 0 to 78%, indicating that temperature directly influences germination (Baskin and Baskin, 1998; Bouwmeester and Karssen, 1992; Probert, 2000). In contrast, seeds of Arum maculatum (Araceae) were dormant, and extensive periods of time at low temperatures are a prerequisite for seed germination. Seeds of A. maculatum failed to germinate over a 1-year period of incubation on agar–water at 16–28 8C but germinated when moved to a lower temperature (Pritchard et al., 1993). A question was whether L. spinosa seeds were induced into dormancy or quiescence at 10 8C? If seeds are quiescent, they are non-dormant but are prevented from germinating due to an unfavorable environmental factor, e.g. a low temperature. In the case of L. spinosa seeds, 10 8C is below the base temperature for germination (Labouriau, 1970; Roberts, 1988). Mohamed et al. (1988) and Moot et al. (2000) reported a similar decline from the optimum to a threshold temperature below which germination ceased. Also, imbibed seeds above the supraoptimal temperature for seed germination are also quiescent (Roberts, 1988), but viability is unlikely to be maintained for a long time at these conditions (Ellis et al., 1987). Previously, the phenomenon of germination failure at low temperature, water stress and poor aeration was called enforced dormancy (Harper, 1977; Baskin and Baskin, 2004). However, Probert (2000) thought these restraints led to quiescence rather than enforced dormancy, since one or more of the three minimum requirements for germination of nondormant seeds is lacking. In the present study, seeds tolerated 60 days at low temperature of 10 8C and were not induced secondary dormancy. Thus, we concluded that L. spinosa seeds were in the state of quiescence or germinated very slowly at 10 8C and deteriorated at 35 8C. Interestingly, some seeds could survive after 6-month cool storage at 10 8C. Judging by the relationship between temperature and germination, we think the most appropriate temperature for wet storage would be 10 8C; this is close to the minimum temperature for germination and thus less germination during storage than at 15 8C. Mata and Moreno-Casasola (2005) found that the germination of Pachira aquatica seeds was not affected by the light environment but the inundation level had a significant effect. Seeds of A. maculatum germinated equally well in light and dark at 11–28 8C (Pritchard et al., 1993), but those of Pistia stratiotes (Araceae)
A.-j. Tang, C.-l. Long / Aquatic Botany 89 (2008) 352–356
germinated to higher percentage in light than in darkness (only 8% in darkness) at 25 8C (Fu, 1957). Thus, sensitivity of Araceae seeds to light depends on the species. In our study, light did not significantly affect the germination percentage of L. spinosa seeds at 25 8C; however, it markedly affected germination rate. Thus, although mature L. spinosa seeds are capable of germinating over a range of conditions, in last 3 years, we have not observed any regeneration from seeds in the field. Natural populations appear to spread mainly by asexual reproduction although plants produce many viable seeds. Recalcitrant seeds pose serious challenges in ex situ germplasm storage as their seeds are intolerant of desiccation and sensitive to chilling (Roberts, 1973). Desiccation and freezing sensitivity affected physiological processes of both seeds and embryonic axes of some recalcitrant seeds, such as tea, cocoa and jackfruit (Chandel et al., 1995), Quercus robur (Hendry et al., 1992), and Acer saccharinum (Pukacka and Ratajczak, 2006). Also, desiccationinduced changes in biochemical and physiological commonly cause loss of viability (Hendry et al., 1992; Smith and Berjak, 1995; Vertucci and Farrant, 1995; Dussert et al., 2006; Berjak, 2006). As far as we know, the cardinal diagnostic feature of recalcitrant seeds is that they cannot be dried without damage, namely, sensitive to desiccation (Roberts et al., 1984; Pammenter and Berjak, 1999). In our study, the moisture loss of L. spinosa seeds decreased exponentially as drying time was extended, indicating that L. spinosa seeds easily lose water under dry stress. Furthermore, L. spinosa seeds were so sensitive to desiccation that only a few seeds (12%) were able to survive 0.13 g H2O g1 dry wt moisture content. Additionally, L. spinosa seeds were sensitive to low temperature (18 8C), and when the storage duration was extended, the seed viability also badly declined at 4 8C. Based on these results, we classified L. spinosa seeds as recalcitrant. As noted by Pammenter and Berjak (2000), seed desiccation sensitivity may be an ancestral state, with tolerance evolving early, and several times independently. Meanwhile, based on a review of 195 sensitive species, Farnsworth (2000) has suggested that the most parsimonious explanation of the current distribution of species’ seed desiccation sensitivity is by convergent loss of tolerance from tolerant ancestors. In our study, considering the aquatic habitats of L. spinosa, with its seeds being shed into very moist environments, we inferred that the seeds may not have evolved desiccation tolerance, making them particularly difficult to maintain in a viable state in long-term storage. Acknowledgements This study was supported by the National Science Research Foundation of China (30170102), and the ministry of Science and Technology of China (2004DKA30430 & 2005DKA21006). Prof. Dr. Song-Quan Song of the Xishuangbanna Tropical Garden (XTBG), Chinese Academy of Sciences, provided necessary support for the laboratory work. Prof. Dr. Greg Welbaum at the Virginia Tech read critically the draft manuscript and proposed helpful suggestions. Also, we are extremely grateful to Ms. Tian Mei-hua (XTBG) and Carol C. Baskin and Jerry M. Baskin (University of Kentucky, Lexington, KY, USA) for their critical reading of the manuscript and brought forward helpful suggestions. References Baskin, C.C., Baskin, J.M., Chester, E.W., 1996. Seed germination ecology of the aquatic winter annual Hottonia inflate. Aquat. Bot. 54, 51–57. Baskin, C.C., Baskin, J.M., 1998. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, San Diego, California, p. 666. Baskin, C.C., Baskin, J.M., 2004. A classification system of seed dormancy. Seed Sci. Res. 14, 1–16.
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Berjak, P., 2006. Unifying perspectives of some mechanisms basic to desiccation tolerance across life forms. Seed Sci. Res. 16, 1–15. Bouwmeester, H.J., Karssen, C.M., 1992. The dual role of temperature in the regulation of seasonal changes in dormancy and germination of seeds of Polygonum persicaria L. Oecologia 90, 88–94. Brenchley, J.L., Probert, R.J., 1996. A discussion of the possible role of seed banks in the conservation of seagrasses. In: Kuo, J., Walker, I.D., Kirkman, H. (Eds.), Seagrass Biology: Scientific Discussion for an International Workshop, Science U.W.A., pp. 65–69. Brenchley, J.L., Probert, R.J., 1998. Seed germination responses to some environmental factors in the seagrass Zostera capricorni from Western Australia. Aquat. Bot. 62, 177–188. Chandel, K.P.S., Chaudhury, R., Radhamani, J., Malik, S.K., 1995. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cacao and jackfruit. Ann. Bot. 76, 443–450. Daws, M.I., Garwood, N.C., Pritchard, H.W., 2005. Traits of recalcitrant seeds in a semi-deciduous tropical forest in Panama´: some ecological implications. Funct. Ecol. 19, 874–885. Daws, M.I., Garwood, N.C., Pritchard, H.W., 2006. Predicting desiccation sensitivity in seeds of woody species: a probabilistic model based on two seed traits and 104 species. Ann. Bot. 97, 667–674. Dussert, S., Davery, M.W., Laffargue, A., Doulbeau, S., Swennen, R., Etienne, H., 2006. Oxidative stress, phospholipids loss and lipid hydrolysis during drying and storage of intermediate seeds. Physiol. Plantarum 127, 192–204. Ellis, R.H., Simon, G., Covell, S., 1987. The influence of temperature on seed germination rate in grain legumes. III. A comparison of five faba bean genotypes using a new screening method. J. Exp. Bot. 38, 1033–1043. Farnsworth, E., 2000. The ecology and physiology of viviparous and recalcitrant seeds. Annu. Rev. Ecol. Syst. 31, 107–138. Harper, J.L., 1977. Population Biology of Plants. Academic Press, London. Fu, J.R., 1957. 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Bot. 39, 1121–1128. Moot, D.J., Scott, W.R., Roy, A.M., Nicholls, A.C., 2000. Base temperature and thermal time requirements for germination and emergence of temperate pasture species. N.Z. J. Agric. Res. 43, 15–25. Pammenter, N.W., Berjak, P., 1999. A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms. Seed Sci. Res. 10, 301–306. Pammenter, N.W., Berjak, P., 2000. Some thoughts on the evolution and ecology of recalcitrant seeds. Plant Species Biol. 2, 153–156. Pons, T.L., 2000. Seed responses to light. In: Fenner, M. (Ed.), Seeds: The Ecology, Regeneration in Plant Communities. 2nd ed. CAB International, Wallingford, pp. 237–260. Pritchard, H.W., Wood, J.A., Manger, K.R., 1993. Influence of temperature on seed germination and the nutritional requirements for embryo growth in Arum maculatum L. New Phytol. 123 (4), 801–809. Pritchard, H.W., Daws, M.I., Fletcher, B.J., Game´ne´, C.S., Msanga, H.P., Omond, W., 2004. Ecological correlates of seed desiccation tolerance in tropical African dryland trees. Am. J. Bot. 91 (6), 863–870. Probert, R.J., 2000. The role of temperature in the regulation of seed dormancy and germination. In: Fenner, M. (Ed.), Seeds: The Ecology and Regeneration in Plant Communities. 2nd ed. CAB International, Wallingford, pp. 261–291. Pukacka, S., Ratajczak, E., 2006. Antioxidative response of ascorbate-glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds. J. Plant Physiol. 163, 1259–1266. Rakondranony, G.L., Sscande, M., Wood, C.B., Pritchard, H.W., 2006. Seed storage responses in four species of threatened genus Ravenea (Araceae). Seed Sci. Technol. 34 (2), 513–517. Roberts, E.H., 1973. Predicting the storage life of seeds. Seed Sci. Technol. 1, 499– 514. Roberts, E.H., King, M.W., Ellis, R.H., 1984. Recalcitrant seeds their recognition and storage. In: Holden, J.H.W., Williams, J.T. (Eds.), Crop Genetic Resources Conservation and Evaluation. George Allen and Unwin, London, pp. 38–52. Roberts, E.H., 1988. Temperature and seed germination. In: Long, S.P., Woodward, F.E. (Eds.), Plant and Temperature. Symposia of the Society of Experimental Biology, vol. 42. Company of Biologists, Cambridge, UK, pp. 109–132. Smith, M.T., Berjak, P., 1995. Deteriorative changes associated with the loss of viability of stored desiccation-tolerant and -sensitive seeds. In: Kigel, J., Galili, G. (Eds.), Seed Development and Germination. Marcel Dekker Inc., New York, pp. 701–746.
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Vertucci, C.W., Farrant, J.M., 1995. Acquisition and loss of desiccation tolerance. In: Kigel, J., Galili, G. (Eds.), Seed Development and Germination. Marcel Dekker Inc., New York, pp. 237–271. Welling, C.H., Pederson, R.L., Van der Valk, A.G., 1988. Recruitment from the seed bank and the development of zonation of emergent vegetation during a drawdown in a prairie wetland. J. Ecol. 76, 483–496.
Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Seed germination of Lasia spinosa as a function of temperature, light, desiccation, and storage An-jun Tang a,b, Chun-lin Long a,* a b
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China Graduate School of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 April 2007 Received in revised form 7 March 2008 Accepted 3 April 2008 Available online 12 April 2008
Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 8C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 8C, after 60 days at 10 8C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 8C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H2O g1 dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H2O g1 dry wt moisture content. Seeds stored at 0.58 g H2O g1 dry wt moisture content at four constant temperatures (4, 10, 15, and 18 8C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H2O g1 dry wt moisture content could be effectively stored for a few months between 10 and 15 8C although the most appropriate temperature for wet storage appears to be 10 8C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 8C. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Lasia spinosa Desiccation tolerance Recalcitrant seeds Seed germination Seed storage Tropical swamp
1. Introduction At present, some tropical wetland species in China are at risk because of deforestation and environmentally unsustainable land use (Tan and Ni, 2006). For example, Lasia spinosa (L.) Thwait. (Araceae), a valuable swamp species which can be used as an ornamental, medicine and vegetable, is decreasing greatly due to excessive extraction and degradation of its habitat (Li, 1979). One method of preserving genetic diversity of this species is to store its seeds in the seed-gene bank. However, before seed storage can be implemented successfully, a full understanding of the requirements for seed germination and desiccation tolerance is essential. Seed sensitivity to desiccation is more common among species growing in permanently moist areas, such as tropical rainforest (Tweddle et al., 2003). In addition, some but not all wetland species produce recalcitrant (desiccation sensitive) seeds (Pammenter and Berjak, 2000), including many riparian species, such as Ravenea
* Corresponding author. Fax: +86 871 5223233. E-mail address: [email protected] (C.-l. Long). 0304-3770/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2008.04.003
rivularis (Arecaceae) (Rakondranony et al., 2006). Finally, some understorey herbs also produce desiccation intolerant seeds (Suzuki et al., 2007). Recalcitrant seeds tend to be produced by trees, are relatively large in size and shed during the rainy season (Pritchard et al., 2004) and germinate relatively rapidly, unhindered by the presence of a relatively thin seed coat (Pritchard et al., 2004; Daws et al., 2005, 2006). Such adaptations increase the likelihood of ensuring continuous moisture, thus avoiding seed desiccation stress (Mata and Moreno-Casasola, 2005). As demonstrated by many studies, seasonal variations in temperature regulate the timing of germination in some aquatic species (Welling et al., 1988; Baskin et al., 1996; Brenchley and Probert, 1996, 1998; Mata and Moreno-Casasola, 2005). Additionally, light significantly affects seed germination of some plants (Pons, 2000). To facilitate successful restoration of swampy ecosystems, information on seed dormancy and germination requirements and storability of seeds of critical species is needed. Thus, we investigated germination characteristics and desiccation sensitivity of L. spinosa seeds. Since L. spinosa is an aquatic species that disperses its seeds into relatively open very moist environments, we hypothesized that seeds may require light for germination and that they may be desiccation intolerant.
A.-j. Tang, C.-l. Long / Aquatic Botany 89 (2008) 352–356
353
2. Materials and methods
2.5. Effect of storage on germination
2.1. Study species and area
Seeds of L. spinosa with a moisture content of 0.58 g H2O g1 dry wt and high viability were stored hermetically at 4, 10, 15, and 18 8C for up to 6 months. After 1, 3 and 6 months, 100 seeds were removed from storage and tested for germination in light at 25 8C, using four replicates of 25 seeds each.
L. spinosa is a perennial evergreen herb growing on wet, nutrientrich soils near or in ditches, brooks, ponds or grass-clusters, commonly characterized by seasonally changing water levels. Such sites are common near villages or on moist farmland in southeastern Asian, including China, Thailand, and Malaya (Li, 1979). Plants of L. spinosa produce one-seeded berries that mature in February. The study was carried out in the Xishuangbanna Tropical Botanical Garden (XTBG) (218410 N, 1018250 E, 570 m altitude) of the Chinese Academy of Sciences. XTBG is located in southern Yunnan province, where annual rainfall is about 1493 mm with a dry season from November to next April. Mean annual temperature is 21.8 8C, and the hottest and coldest months are June and January, respectively, with a mean temperature of 25.7 and 16 8C, respectively (http://cbis.xtbg.ac.cn/db/PDF_LYR/476265.pdf). 2.2. Seed collection and storage To obtain a sufficient quantity of seeds, fruits were collected from 100 plants growing in the XTBG on 6 February 2004. Within 1 day after collection, seeds were separated carefully from the berries by hand, cleaned and stored in paper bags at room temperature (20 2 8C) for 1 or 2 days, except for those used to determine initial moisture content. Also, we measured the dimension (length breadth) of 100 seeds and the 1000-seed weight. 2.3. Germination requirements
2.6. Data analysis Percentage germination was calculated for all tests. Analysis of variance was used to determine the effects of factors on germination percentage (arcsine transformed for linearity). Differences between means were determined using least significant difference (LSD), or standard errors of the mean values. 3. Results 3.1. Germination requirements Optimal temperature for germination (97%) was 25 8C, but seeds also germinated to 80% at 30 8C (Fig. 1). Daily fluctuation of 20/30 8C was also effective for a high germination response (94% germination) (Fig. 1). Below 20 8C, percentage germination was very low, and no germination occurred at 10 8C even after 60 days of incubation. When seeds were transferred from 10 to 25 8C, they germinated to 17 3.2%, 29 4.7% and 78 2.8% after 5, 15 and 20 days, respectively. Freshly harvested seeds germinated to 97% and 86% at 25 8C in light and in dark, respectively (Fig. 2). Although final
Four replicates of 25 freshly collected seeds each were placed in 9 cm diameter glass Petri dishes on filter paper moistened with distilled water and placed incubators equipped with a cool, white fluorescent light (HPG-280B, Har’erbin, China) providing a photosynthetic photon flux density (PPFD) measured with LI-1400 Data logger (LI-COR, USA) of approximately 44 mmol m2 s1 over the waveband 400–700 nm. Seeds were exposed to a daily 14 h daily photoperiod at constant temperatures of 10, 15, 20, 25, 30, 35, and a daily 14 h fluctuating cycle of 20/30 8C. Germination counts were made daily for 30–80 days depending on germination rate in relation to temperature. At the end of a 60-day incubation period at 10 8C, seeds that had not germinated were transferred to 25 8C for 20 days. In addition, we compared germination under light and continuous dark at 25 8C using a green safe light to examine seeds incubated in darkness. Distilled water was added as required to ensure that moisture was non-limiting for germination. Seeds were recorded as germinated when the protruding radicle was 2 mm in length. 2.4. Desiccation sensitivity Moisture content for freshly collected seeds was calculated immediately after cleaning according to recommended protocols of the International Seed Testing Association (ISTA, 1999). During slow dehydration, seeds were spread in a monolayer and dried for up to 3 weeks in a dry room operating at 15 8C and 45% relatively humidity (RH). Moisture contents of all seed lots were determined gravimetrically before and after drying in a 103 8C oven for 17 h (ISTA, 1999). At each sample time during the drying curve, we withdrew 15 seeds to determine seed moisture content. Seed moisture content was expressed on the dry weight basis (i.e., g H2O g1 dry wt). Each time seed moisture was determined, seeds were tested for germination in light at 25 8C, using four replicates of 25 seeds each.
Fig. 1. Effects of temperature on germination of Lasia spinosa seeds. Bars indicate standard deviations.
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A.-j. Tang, C.-l. Long / Aquatic Botany 89 (2008) 352–356 Table 1 Effects of storage temperature and duration on viability of Lasia spinosa seeds with 0.58 g H2O g1 dry wt moisture content Storage period (months)
Germination (%) (S.D.) after storage 4 8C
10 8C
15 8C
18 8C
1 3 6
83 0.58a 37 2.83b 18 1.42c
86 2.2a 76 0.8a 74 1.5a
84 3.8a 81 0.7a 77 1.3a
0 0 0
Germination percentage is the mean value of four replicates 1 S.D. For each storage period, differences between temperatures (P < 0.05) are indicated by different lowercase letters. In each column values followed by the same letter are not significantly different (P < 0.05).
Fig. 2. Effect of light (14 h daily photoperiod) and continuous darkness on germination of Lasia spinosa seeds at 25 8C.
declined gradually, with an increase in storage duration. The greatest decrease in viability (from 83% to 18%) occurred at 4 8C (Table 1). Seed viability was not significantly reduced during storage at 10 or 15 8C, suggesting that prolonging storage duration would not impair seed viability at 10 or 15 8C. 4. Discussion
germination percentages in the light and dark did not significantly differ (P > 0.05), dark germination took more than twice as long as that in light (Fig. 2). 3.2. Desiccation sensitivity L. spinosa seeds were relatively large and heavy, with a mean dimension of 100 seeds of 1.1 cm 0.8 cm (length breadth) and a 1000-seed weight of 306.4 2.2 g. Water content of L. spinosa seeds sharply decreased with drying time at 15 8C and 45% RH (Fig. 3). As seed moisture content decreased from 2.02 to 0.58 g H2O g1 dry wt, seed germination percentage remained high (Fig. 3). However, further drying to 0.13 g H2O g1 dry wt caused almost complete loss of viability. The critical moisture for viability loss, i.e., the mid-point, was about 0.35 g H2O g1 dry wt (or 26% moisture content, fresh weight basis). 3.3. Effect of storage on germination Storage duration combined with temperature had strong effects on the germination of L. spinosa seeds with 0.58 g H2O g1 dry wt moisture content (Table 1). No germination occurred after 1, 3 or 6 months of storage at 18 8C. At 4, 10 and 15 8C, seed viability
Fig. 3. Relationship between moisture content of Lasia spinosa seeds and drying time at 15 8C and 45% RH (*) and effects of moisture content on germination of L. spinosa seeds at 25 8C (*). Bars indicate standard deviations.
Freshly harvested mature seeds of L. spinosa seeds were nondormant and germinated to high percentages at 25 and 30 8C. When seeds were transferred from 10 to 25 8C after 60 days, germination increased from 0 to 78%, indicating that temperature directly influences germination (Baskin and Baskin, 1998; Bouwmeester and Karssen, 1992; Probert, 2000). In contrast, seeds of Arum maculatum (Araceae) were dormant, and extensive periods of time at low temperatures are a prerequisite for seed germination. Seeds of A. maculatum failed to germinate over a 1-year period of incubation on agar–water at 16–28 8C but germinated when moved to a lower temperature (Pritchard et al., 1993). A question was whether L. spinosa seeds were induced into dormancy or quiescence at 10 8C? If seeds are quiescent, they are non-dormant but are prevented from germinating due to an unfavorable environmental factor, e.g. a low temperature. In the case of L. spinosa seeds, 10 8C is below the base temperature for germination (Labouriau, 1970; Roberts, 1988). Mohamed et al. (1988) and Moot et al. (2000) reported a similar decline from the optimum to a threshold temperature below which germination ceased. Also, imbibed seeds above the supraoptimal temperature for seed germination are also quiescent (Roberts, 1988), but viability is unlikely to be maintained for a long time at these conditions (Ellis et al., 1987). Previously, the phenomenon of germination failure at low temperature, water stress and poor aeration was called enforced dormancy (Harper, 1977; Baskin and Baskin, 2004). However, Probert (2000) thought these restraints led to quiescence rather than enforced dormancy, since one or more of the three minimum requirements for germination of nondormant seeds is lacking. In the present study, seeds tolerated 60 days at low temperature of 10 8C and were not induced secondary dormancy. Thus, we concluded that L. spinosa seeds were in the state of quiescence or germinated very slowly at 10 8C and deteriorated at 35 8C. Interestingly, some seeds could survive after 6-month cool storage at 10 8C. Judging by the relationship between temperature and germination, we think the most appropriate temperature for wet storage would be 10 8C; this is close to the minimum temperature for germination and thus less germination during storage than at 15 8C. Mata and Moreno-Casasola (2005) found that the germination of Pachira aquatica seeds was not affected by the light environment but the inundation level had a significant effect. Seeds of A. maculatum germinated equally well in light and dark at 11–28 8C (Pritchard et al., 1993), but those of Pistia stratiotes (Araceae)
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germinated to higher percentage in light than in darkness (only 8% in darkness) at 25 8C (Fu, 1957). Thus, sensitivity of Araceae seeds to light depends on the species. In our study, light did not significantly affect the germination percentage of L. spinosa seeds at 25 8C; however, it markedly affected germination rate. Thus, although mature L. spinosa seeds are capable of germinating over a range of conditions, in last 3 years, we have not observed any regeneration from seeds in the field. Natural populations appear to spread mainly by asexual reproduction although plants produce many viable seeds. Recalcitrant seeds pose serious challenges in ex situ germplasm storage as their seeds are intolerant of desiccation and sensitive to chilling (Roberts, 1973). Desiccation and freezing sensitivity affected physiological processes of both seeds and embryonic axes of some recalcitrant seeds, such as tea, cocoa and jackfruit (Chandel et al., 1995), Quercus robur (Hendry et al., 1992), and Acer saccharinum (Pukacka and Ratajczak, 2006). Also, desiccationinduced changes in biochemical and physiological commonly cause loss of viability (Hendry et al., 1992; Smith and Berjak, 1995; Vertucci and Farrant, 1995; Dussert et al., 2006; Berjak, 2006). As far as we know, the cardinal diagnostic feature of recalcitrant seeds is that they cannot be dried without damage, namely, sensitive to desiccation (Roberts et al., 1984; Pammenter and Berjak, 1999). In our study, the moisture loss of L. spinosa seeds decreased exponentially as drying time was extended, indicating that L. spinosa seeds easily lose water under dry stress. Furthermore, L. spinosa seeds were so sensitive to desiccation that only a few seeds (12%) were able to survive 0.13 g H2O g1 dry wt moisture content. Additionally, L. spinosa seeds were sensitive to low temperature (18 8C), and when the storage duration was extended, the seed viability also badly declined at 4 8C. Based on these results, we classified L. spinosa seeds as recalcitrant. As noted by Pammenter and Berjak (2000), seed desiccation sensitivity may be an ancestral state, with tolerance evolving early, and several times independently. Meanwhile, based on a review of 195 sensitive species, Farnsworth (2000) has suggested that the most parsimonious explanation of the current distribution of species’ seed desiccation sensitivity is by convergent loss of tolerance from tolerant ancestors. In our study, considering the aquatic habitats of L. spinosa, with its seeds being shed into very moist environments, we inferred that the seeds may not have evolved desiccation tolerance, making them particularly difficult to maintain in a viable state in long-term storage. Acknowledgements This study was supported by the National Science Research Foundation of China (30170102), and the ministry of Science and Technology of China (2004DKA30430 & 2005DKA21006). Prof. Dr. Song-Quan Song of the Xishuangbanna Tropical Garden (XTBG), Chinese Academy of Sciences, provided necessary support for the laboratory work. Prof. Dr. Greg Welbaum at the Virginia Tech read critically the draft manuscript and proposed helpful suggestions. Also, we are extremely grateful to Ms. Tian Mei-hua (XTBG) and Carol C. Baskin and Jerry M. Baskin (University of Kentucky, Lexington, KY, USA) for their critical reading of the manuscript and brought forward helpful suggestions. References Baskin, C.C., Baskin, J.M., Chester, E.W., 1996. Seed germination ecology of the aquatic winter annual Hottonia inflate. Aquat. Bot. 54, 51–57. Baskin, C.C., Baskin, J.M., 1998. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, San Diego, California, p. 666. Baskin, C.C., Baskin, J.M., 2004. A classification system of seed dormancy. Seed Sci. Res. 14, 1–16.
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