Abstract
Kapok (Bombax ceiba) is a deciduous tree that can grow in the dry-hot valley of southwestern China where its natural regeneration by seedlings is difficult. As mature fruit split open and seeds disperse by wind, it is difficult to collect fully mature seeds. The effects of seed moisture content (MC) and storage temperatures on seed germination of dark-brown seeds collected from split fruit and light-brown seeds collected ≈10–15 days earlier than the time of fruit split were studied to determine the effective germplasm preservation via the seeds. Dark-brown mature seeds could tolerate desiccation to less than 5% MC and could tolerate −20 and −80 °C. Seeds of kapok showed orthodox storage behavior. They can be stored at subzero temperatures with low MC for a long time. For light-brown seeds, germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) decreased significantly after seed desiccation. Germination percentage of light-brown seeds with different MC increased to a different extent after being stored at different temperatures for 1 year (76% to 99%), compared with the fresh seeds (73%). Storing fresh seeds at 4 °C was most favorable to keep seed viability and seed vigor of light-brown seeds. Seed collection could be done several days earlier than the time of fruit burst to ensure increased quantity of collected seeds.
Kapok is a tall tree buttressed at the base, widely found throughout India and other parts of tropical and subtropical Asia, Australia, and Africa (Flora of China Editorial Committee, 1984; Verma et al., 2011). Kapok can grow in the southwestern China where the adult trees of the species produce many seeds; however, few seedlings are found around the mature trees (Zheng et al., 2013; Zheng and Ma, 2014). The research by Ballabha et al. (2013) also showed poor regeneration of kapok in India. Koenig and Griffiths (2012) reported that seeds of kapok did not survive in the soil past the wet season to form a seed bank. Therefore, germplasm preservation through seed storage is necessary for kapok. Understanding storage behavior of seeds is essential to determine the most suitable storage environment, and the likely duration of successful storage (Hong and Ellis, 1996). However, the storage behavior of this species has not previously been studied in detail. Although Nakar and Jadeja (2014) suggested that seeds of kapok were recalcitrant type, their classification was simply based on seed viability. The Seed Information Database [SID (Royal Botanic Gardens, Kew, 2015)] lists it as “probably orthodox.”
In the wild, kapok grows to an average of 20 m tall, with old trees up to 60 m in wet tropical weather. The brown, mature fruit of kapok split open on the tree and the seeds disperse by wind making it difficult to collect fully mature seeds. Generally, unsplit fruit, which can be brown or green can be collected and placed at room temperature and seeds can be collected once the fruit burst. However, based on several years of our observation, the color of these seeds is lighter (light brown) than that of the normal mature seeds (dark brown), which are collected after the fruit split on the trees. We suspect that light-brown and dark-brown seeds may possess different maturity and thus might show different physiological responses to desiccation and subzero temperatures. In practice, most of the collected kapok fruit are the unsplit fruit. So far, the effects of storage on seed germination of light-brown seeds are not clear, and this is a disadvantage to the reforestation and restoration projects in the region.
In the present study, the effects of desiccation and storage temperature on seed germination of dark-brown and light-brown seeds in kapok were tested to determine the seed storage behavior and to determine if dark-brown and light-brown seeds possess different sensitivity to desiccation and low temperatures. The results can provide guidelines for seed storage and seed collection timing.
Materials and methods
Fruit and seeds of kapok were collected from Lujiangba, Baoshan, Yunnan Province (lat. 24°54′N, long. 98°53′E) before fruit burst in Apr. 2012 (≈10–15 d earlier than the time of fruit burst) and after fruit burst in May 2013. Lujiangba is in the south subtropical dry and hot river valley areas. Mean temperature of the coldest and warmest months are 14.7 and 26.3 °C, respectively. The mean annual precipitation is 755 mm, with most rainfall concentrated between June and October (Li et al., 2009). The unsplit fruit were put at room temperature until they split open. Seeds were selected from the split fruit manually and used immediately for the experiment. The color of seeds collected in April (light brown) was obviously lighter than that of normal mature seeds collected in May, which were dark brown.
Desiccation tolerance was compared between light-brown and dark-brown seeds. To determine seed tolerance to desiccation, light-brown and dark-brown seeds were dried to different MCs in sealed containers with silica gel at room temperature (mean temperature in Apr. 2012 and May 2013 of 20 ± 2 °C). Seeds were removed and MC and germination were tested after 0, 1, 2, and 5 d of drying for light-brown seeds and 0, 3, and 5 d of drying for dark-brown seeds. Moisture content of 20 seeds in four replications was determined on a fresh weight basis after oven drying for 17 h at 103 °C. To determine the effects of seed MC on seed viability and seed vigor of light-brown seeds stored at different temperatures, seeds desiccated for 0, 1, 2, and 5 d were sealed in aluminum foil bags and stored at room temperature (mean annual temperature of 14.7 °C), 4, and −20 °C for 1 year, respectively. To determine the tolerance of dark-brown seeds to low temperature, seeds desiccated for 5 d were sealed in aluminum foil bags and stored at −20 and −80 °C for 3 months. The germination characteristics of fresh dark-brown seeds were used as the control. Germination was determined at the end of each storage time.
Statistical analyses were performed using SPSS 15.0 (IBM Corp., Armonk, NY). Germination percentage was subjected to arcsin transformation before statistical analysis. GP, GI, SFW, and VI of seeds desiccated for different periods or stored at different temperatures were subjected to one-way analysis of variance for dark-brown and light-brown seeds, respectively. Significant differences between means were tested by Fisher’s least significant difference at 0.05 level of probability.
Results and discussion
Moisture content of dark-brown mature (collected May 2013) seeds was 11.10% and it decreased to 6.35% and 4.98% after 3 and 5 d of desiccation, respectively. GP, GI, SFW, and VI of the fresh dark-brown seeds were 92.00%, 7.50, 0.26 g, and 1.92, respectively. Desiccation did not significantly affect germination-related indexes mentioned above (Fig. 1). It showed that dark-brown mature seeds were tolerant to desiccation. After 3 months storage at −20 and −80 °C, GP and SFW of mature seeds with MC of 4.98% did not change significantly compared with the control. However, GI decreased significantly by 26.67% and 29.33%, respectively, and VI also decreased significantly by 30.73% and 35.42%, respectively (Fig. 2). GI reflects the rate of seed germination and its decrease was mainly due to the slow metabolic rate of seeds that were immediately retrieved from storage at subfreezing temperatures. As SFW did not decrease, decrease of VI also occurred because of the slow metabolic rate of seeds.
The effect of desiccation on germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of dark-brown mature seeds (collected from split fruit in 2013) of kapok. The moisture content of seeds desiccated for 0, 3, and 5 d was 11.10%, 6.35%, and 4.98%, respectively. Data points represent means of four replicates ±sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
The effect of desiccation on germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of dark-brown mature seeds (collected from split fruit in 2013) of kapok. The moisture content of seeds desiccated for 0, 3, and 5 d was 11.10%, 6.35%, and 4.98%, respectively. Data points represent means of four replicates ±sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
The effect of desiccation on germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of dark-brown mature seeds (collected from split fruit in 2013) of kapok. The moisture content of seeds desiccated for 0, 3, and 5 d was 11.10%, 6.35%, and 4.98%, respectively. Data points represent means of four replicates ±sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of dark-brown mature seeds (collected from split fruit in 2013) of kapok with moisture content of 4.98% after storage at −20 and −80 °C for 3 months. Data points represent means of four replicates ± sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; (1.8 × °C) + 32 = °F, 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of dark-brown mature seeds (collected from split fruit in 2013) of kapok with moisture content of 4.98% after storage at −20 and −80 °C for 3 months. Data points represent means of four replicates ± sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; (1.8 × °C) + 32 = °F, 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of dark-brown mature seeds (collected from split fruit in 2013) of kapok with moisture content of 4.98% after storage at −20 and −80 °C for 3 months. Data points represent means of four replicates ± sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; (1.8 × °C) + 32 = °F, 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Three categories of seed storage behavior have been identified: orthodox, intermediate, and recalcitrant (Hong and Ellis, 1996). Orthodox seeds can be dried to a MC of 5% or less (fresh weight basis) and stored at subzero temperatures for a long period. On the other hand, recalcitrant seeds cannot be dried to a MC less than 15% to 30%, depending on species, or stored at subzero temperatures without losing viability. Intermediate seeds can tolerate drying to ≈7% to 12% MC, but further drying and storage at subzero temperatures; e.g., −20 °C, reduces seed longevity (Chen et al., 2008; Hong and Ellis, 1996). On the basis of the tolerance of mature seeds to desiccation and subzero temperatures, seeds of kapok are orthodox seeds (Hong and Ellis, 1996). However, Nakar and Jadeja (2014) suggested that seeds of kapok were recalcitrant type. As the classification adopted by Nakar and Jadeja (2014) was simply based on seed viability (lack of seed dormancy) not on the seed sensitivity to desiccation and low temperatures, we think their conclusion was arbitrary. Seed potential longevity is affected by genotype and provenance. And how much of the potential longevity is realized depends on subsequent storage conditions (Hong and Ellis, 1996). Species of trees with orthodox seed storage characteristics can be stored for long periods and up to hundreds of years in some cases (Bewley et al., 2013; Simpson et al., 2004). Thus, seeds of kapok can be stored at subzero temperatures with low MC for a long time. In the wild, seeds of kapok did not survive in the soil past the wet season to form a seed bank (Koenig and Griffiths, 2012) and the regeneration of this species is poor (Ballabha et al., 2013; Zheng et al., 2013). Thus, seed storage is an effective method for long-term conservation of the genetic resources of kapok.
Moisture content of light-brown seeds was 12.8% and it decreased to 7.48%, 5.51%, and 4% after 1, 2, and 5 d of desiccation, respectively. GP, GI, SFW, and VI of fresh light-brown seeds were 73%, 5.58, 0.27 g, and 1.53, respectively. The corresponding GIs decreased significantly with the decreasing of seed MC, decreasing by 65.75%, 76.70%, 51.85%, and 88.89%, respectively, for the seeds with MC of 4% (Fig. 3). This indicated that light-brown seeds of kapok were sensitive to desiccation and may not be fully mature. As mature seeds of kapok are tolerant to desiccation but the light-brown (immature) seeds are not. The desiccation tolerance of kapok seeds appears to be related to seed maturity. Similar results have also been reported in many plants, in which immature seeds/embryos are desiccation sensitive and mature seeds/embryos are desiccation tolerant (Huang et al., 2011; Kalemba et al., 2009). It has been reported that at later stages of seed development such as at the end of the seed-filling phase many orthodox species may still show damage on desiccation to very low MC levels (Ellis and Hong, 1994; Hong and Ellis, 1996). It has been well studied that orthodox seeds undergo maturation drying on the mother plant and a number of mechanisms are involved in the acquisition of desiccation tolerance, such as antioxidant systems, late embryogenesis–abundant proteins, carbohydrates, and subcellular organization (Connor and Sowa, 2003; Farrant et al., 1997).
The effect of desiccation on seed germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of light-brown of kapok (collected ≈10–15 d earlier than the time of fruit burst in 2012). The moisture content of seeds desiccated for 0, 1, 2, and 5 d was 12.80%, 7.48%, 5.51%, and 4.00%, respectively. Data points represent means of four replicates ± sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
The effect of desiccation on seed germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of light-brown of kapok (collected ≈10–15 d earlier than the time of fruit burst in 2012). The moisture content of seeds desiccated for 0, 1, 2, and 5 d was 12.80%, 7.48%, 5.51%, and 4.00%, respectively. Data points represent means of four replicates ± sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
The effect of desiccation on seed germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of light-brown of kapok (collected ≈10–15 d earlier than the time of fruit burst in 2012). The moisture content of seeds desiccated for 0, 1, 2, and 5 d was 12.80%, 7.48%, 5.51%, and 4.00%, respectively. Data points represent means of four replicates ± sd. The bars indicated by different lowercase letters are significantly different by Fisher’s least significant difference at P < 0.05; GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Light-brown seeds with different MC were stored at different temperatures for 1 year. For seeds stored at room temperature, GP did not vary significantly among seeds with different MC [total average 90.75% (Fig. 4)]. GI (mean 4.76) and VI (mean 0.99) of seeds with MC of 4% were significantly higher than that of seeds with higher MC. SFW of seeds with MC of 4% was significantly higher than that of seeds with MC of 12.8%. For seeds stored at −4 °C, GP did not vary significantly among seeds with different MC (total average 96.75%). GI (mean 8.78) and VI (mean 2.02) of seeds with MC of 12.8% were significantly higher than that of seeds with relatively low MC. SFW of seeds with MC of 12.8% (mean 0.23 g) was significantly higher than that of seeds with MC of 4% (mean 0.20 g). For seeds stored at −20 °C, GP of seeds with MC of 12.8% and 7.48% (92% and 89%, respectively) was significantly higher than that of seeds with MC of 5.51% and 4% (78% and 76%, respectively). GI (mean 5.53), and VI (mean 1.33) of seeds with MC of 12.8% were significantly higher than that of seeds with relatively low MC. SFW did not vary significantly among seeds with different MC (total average 0.23 g). This showed that relatively high seed MC was more conducive to maintain seed viability for seeds stored at 4 and −20 °C and storing fresh seeds at 4 °C was most favorable to keep seed viability and seed vigor. Studies on other species have also suggested that there is an optimum water content for each storage temperature, which increases as storage temperature decreases (Buitink et al., 2000; Vertucci et al., 1994). Compared with the fresh seeds, GP of kapok seeds with different MCs increased to different extents after being stored for 1 year. Therefore, light-brown seeds of kapok collected from unsplit fruit on the trees can be stored to keep their germinability. This is important in practice to the reforestation and restoration projects. Our laboratory work also showed that seed viability and seed vigor of kapok seeds with high MC (collected from unsplit fruit) were improved after some days of slow-rate desiccation at room temperatures (data not shown). It is probable that fresh light-brown seeds of kapok may exhibit dormancy characteristics, and some changes including physiological processes as well as gene expression and metabolic programs may occur during the period of desiccation and storage. This needs further work to be confirmed.
Seed germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of light-brown seeds (collected ≈10–15 d earlier than the time of fruit burst in 2012) of kapok with different moisture content after storage at different temperatures for 1 year. The moisture content of seeds desiccated for 0, 1, 2, and 5 d was 12.80%, 7.48%, 5.51%, and 4.00%, respectively. Data points represent means of four replicates ± sd. GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; (1.8 × °C) + 32 = °F, 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Seed germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of light-brown seeds (collected ≈10–15 d earlier than the time of fruit burst in 2012) of kapok with different moisture content after storage at different temperatures for 1 year. The moisture content of seeds desiccated for 0, 1, 2, and 5 d was 12.80%, 7.48%, 5.51%, and 4.00%, respectively. Data points represent means of four replicates ± sd. GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; (1.8 × °C) + 32 = °F, 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
Seed germination percentage (GP), germination index (GI), seedling fresh weight (SFW), and vigor index (VI) of light-brown seeds (collected ≈10–15 d earlier than the time of fruit burst in 2012) of kapok with different moisture content after storage at different temperatures for 1 year. The moisture content of seeds desiccated for 0, 1, 2, and 5 d was 12.80%, 7.48%, 5.51%, and 4.00%, respectively. Data points represent means of four replicates ± sd. GI = ∑(Gt/Dt), where Gt is the number of seeds germinating on the tth day of incubation and Dt is the number of days of seed incubation; VI = GI × SFW; (1.8 × °C) + 32 = °F, 1 g = 0.0353 oz.
Citation: HortTechnology hortte 26, 1; 10.21273/HORTTECH.26.1.83
For many species, mature seeds are used to store germplasm and propagate seedlings. However, for some species with dormant seeds, higher frequencies of germination have been achieved by culturing immature seeds rather than by culturing mature seeds (Light and MacConaill, 1998). It has been postulated that dormancy is induced by accumulation of some inhibitory substances or by increasing impermeability of the embryos during seed maturation (van der Kinderen, 1987; Yamazaki and Miyoshi, 2006). For kapok, as mature fruit split open and seeds disperse by wind, it is difficult to collect fully mature seeds. In addition, as light-brown seeds of kapok have relatively high GP and appropriate storage conditions could improve the seed viability and seed vigor, light-brown seeds could be collected several days earlier than the time of fruit burst on the trees to increase the quantity of collected seeds.
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