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Introduction
The genus Daphne includes 44 species of evergreen shrubs distributed in Northwestern China, ranging from Sichuan and Shanxi to Gansu provinces. Daphne giraldii Nitsche (Thymelaeaceae) is a slow-growing shrub with a maximum height of 2 m and has flexible and brown stems. It often grows in virgin land on hillsides and occasionally in ravines, grassland, forest edges and thickets at elevations of 2,400 m to 2,500 m in the Qilian Mountains of China (Zhao, 2007). Daphne giraldii has great ornamental value because of its beautiful yellow flowers, dark green leaflets and red ball-shaped fruits with shells. The plant is cold tolerant and alkali-resistant and shows great adaptation to the environments in Northwestern China and it is used for landscape design. Furthermore, the dried stem and root bark of D. giraldii are known as ''ZuShima'' (Zhao, Jin, & Zhang, 2012), which are commonly used in traditional Chinese medicine for the treatment of aches and rheumatism, particularly for rheumatoid arthritis (Li, Wu, & Yin, 2002). Because of the increasing consumption of D. giraldii, it is necessary to produce this evergreen shrub by developing methods for its propagation. Daphne giraldii propagation is mainly done by cuttings and seeds, but cuttings have lower survival and seeds are submitted to dormancy (Wang, Yan, & Mao, 2012), which is commercially efficient and therefore not recommended for large-scale propagation. Seeds of many shrub species cannot germinate even if we have optimal moisture, oxygen, and soil conditions (Mark, Tony, & Andrew, 2012). This phenomenon is called dormancy, which is quite important in the wild and it is an adaptive mechanism that ensures the survival of some species through periods of environmental stress (Baskin & Baskin, 2004; Gusano, Gomez, & Dicenta, 2004; Kermode, 2005); however, dormant seeds require treatment prior to planting; pre-sowing treatments such as cold stratifications and seed burial (Merritt, Turne, Clarke, & Dixon, 2007; Travlos, Economou, & Karamanos, 2007) have been used to reduce seed hardness and to improve germination and emergence rate. Chemical plant growth regulators such as 6-benzyladenine (6-BA) and exogenous gibberellic acid (GA3) play an important role in dormancy release and in the promotion of germination by increasing the growth potential of the embryo (Kucera, Cohn, & Leubner, 2005; Siddiqui, Mujib, & Maqsood, 2011). Chemical plant growth regulator, stratifications temperature and seed burial are three factors that can potentially affect seed dormancy and germination. Many researchers studied the influence of these factors and found that Diren (Melastoma dodecandrum Lour) seeds soaked in 250-1000 mg·L-1 GA3 or 50-200 mg·L-1 6-BA for 24 h can significantly increase its germination percentage and germination index (Tang, Wei, Yang, Liang, & Wei, 2012). Different seed species need different stratification temperature (-5~10 °C) and time (30-150 d) for dormancy breaking (Walck & Hidayati, 2004; Walck, Hidayati, & Okagami, 2002; Tang et al., 2012); Artemisia ordosica Krasch. and Ceratocarpus arenarius L. seeds buried at a depth of 2-12 cm in sand or peat soil could effectively improve the seed germination percentages (Liu, Zhang, Yin, & Zhang, 2013; Wolfgang, 2002). Seedling emergence and successful establishment of a plant population are mainly regulated by dormancy breaking. Seed dormancy could therefore be an important limiting factor for D. giraldii propagation, since a successful method and technique to overcome seed dormancy in D. giraldii has not been reported. The aims of this study were to improve seed germination in nursery production of D. giraldii seedlings or in seeds sown in the field for large-scale use and to find the most efficient method to break the seed dormancy in order to accelerate its seed germination. Specifically, we studied the effects of various dormancy-breaking treatments (submersion in GA3, 6-BA, cold stratification and burial seed) on seed germination performance of D. giraldii.
Materials and methods
D. giraldii seed collection and storage conditions
Freshly matured fruits consisting of a nutlet (hereafter called seeds) encapsulated by red flesh were collected on August 20, 2013. The fruits were obtained from the elevation (2,450 m) of Buer township in the Qilian Mountains with the following geographical coordinates: latitude 36° 43' North and longitude 97° 25' East with relatively warm summers and cold winters located in the Northwestern part of China. The seeds were separated from the fruit and dried after 6 days in the laboratory (20 °C to 22 °C). Then the seeds were cleaned and placed in paper bags at 25 ± 1 °C and were kept in the laboratory until use.The seeds used for chemical treatment and stratification treatment remain stored for about 8 d, the seeds used for different seed burial experiment remain stored for 35-75 d. The seeds of D. giraldii were 5 to 6 mm in diameter, ball-shaped and brown.
Effect of chemical treatment on dormancy of D. giraldii
Prior to experimental treatment, the collected seeds were examined to remove those that were stained, discolored, and damaged. All seeds were bathed in water, and floating seeds were removed from the water surface. Seeds were sterilized by immersion in 75 % alcohol for 30 min and rinsed three times using distilled water. Then the seed coats were removed by breaking the shell without damaging the endosperm and embryo. The experiment was therefore conducted as follows: (1) The seeds were immersed in 100, 150, 200 and 250 mg·L-1 GA3 solution for 8, 16 and 24 h, (2) The seeds were soaked in 15, 30, 45 and 60 mg·L-1 6-BA solution for 8, 16 and 24 h, (3) The seeds were soaked for 8, 16 and 24 h using the following combinations: 100 mg·L-1 GA + 15 mg·L-1 6-BA, 150 mg·L-1 GA3 + 30 mg·L-1 6-BA, 200 mg·L-1 GA3 + 45 mg·L-1 6-BA, and 250 mg·L-1 GA3 + 60 mg·L 6-BA. Distilled water was used for the controls, three replications of 50 seeds were prepared for each treatment. After treatments, the seeds were placed in 9 cm-diameter Petri dishes with a filter paper moistened with 5 mL of distilled water at a day/ night temperature of 25/22 °C in a 12-h photoperiod supplied by fluorescent lights (70-110 µmol·m-2·s-1, 400-600 nm) and 50 % relative humidity. The Petri dishes were observed daily until the onset of germination. At the end of the experiment, the seeds were considered germinated if the radicle reached 1 mm in length. Germination was recorded daily until its cessation with a minimum of 10 d. Germination percentage (GP) and rates of germination (GR) were calculated according to the following formula (Olmez, Goktur, & Temel, 2007):
GP = n/m *100
where:
n = Number of germinated seeds
m = Number of viable seeds initiated.
GR = [(n1* v1) + (n2 * v2) + (ni * vi)] / M
where:
ni = Number of days for each counting
vi = Number of germinated seeds in each countin.
M = Total number of germinated seeds.
Effect of incremental stratification on dormancy of D. giraldii
D. giraldii seeds were placed into glass Petri dishes and mixed with moist sand (seeds: sand =1:6, v/v). The moisture of the sand and seeds was examined continuously to avoid drying and poor aeration. Triplicate Petri dishes of 50 seeds were prepared and placed into an incubator in the dark at -5 °C, 0 °C, 5 °C and 10 °C for 30, 45, 60, 70 and 90 days. After stratifying the seeds for various durations at various temperatures, germination tests were conducted.
Effect of seed burial on dormancy of D. giraldii
On October 1 and 21, and on November 11, 2013, three packages of replicates of 50 seeds were placed separately into fine-mesh nylon bags. These seeds were buried in a sandy loam from the Hexi University, Zhangye with burial depth increments of 20, 40, 60, 80, 100 and 120 cm. No supplemental irrigation was provided. Treatment packages were recovered on April 10, 2014 (The seeds for the former three burial treatment remain in the soil for about 190, 170 and 150 days, respectively), and the seeds were placed in 9-cm diameter Petri dishes with a filter paper moistened with distilled water. The control was the cleaned seeds stored in paper bags at 25 ± 1 °C in the laboratory. Germination tests were conducted as previously described.
Statistical analyses
ANOVA and Duncan's multiple range tests were carried out to analyze the data (Duncan, 1955) and the pre- treatment effects on GP and GR were determiden using the SPSS 10.0 software package (Statistical Package for the Social Sciences [SPSS], 2000). GP data were arcsine transformed before analysis. Tukey multiple comparison test (LSD test) was performed to determine the treatments with significant differences (P = 0.05). GP and GR obtained in tests on chemical treatment were analyzed by means of a three-factorial ANOVA, whereas the data obtained from the other treatment were analyzed by means of a two-factorial ANOVA. The figures were created with Origin 8.0 (OriginLab Corporation, 2007) when ANOVA indicated significant treatment effects (P ≤ 0.05).
Results and discussion
Effect of chemical treatment on dormancy of D. giraldii
Figures 1 and 2 show GP (over a 10 day period) and GR of various chemically-treated seeds. The different chemical treatments were effective. GP and GR of fresh seeds were increased by GA3 treatment (Figures 1 and 2). GP of seeds treated with 100,150, 200 and 250 mg·L-1 GA3 for 16 h reached 17.3, 15.8, 15.1 and 14.7 %, respectively. Whereas the highest GR of those seeds treated with the same solutions and hours (16 h) reached 0.61 (SD = 0.03). All treatments were significantly different from the control seeds treated with distilled water (P < 0.05). There is a similar trend of variation of GP and GR by GA3 treatment for 8 h and 24 h. Statistical analyses showed that 6-BA treatment could also increase GP and GR significantly compared to the control (P < 0.05), those seeds soaked in 6-BA (30 mg·L-1) solutions for 16 h germinated to a maximum of 17.11 % (SD = 1.15, Figure 1), slightly lower than the maximum of GA3 treatment (17.3 %, SD = 0.75, Figure 1), but there are no obvious difference between them (P > 0.05). Among all the chemical treatments, the highest GP and GR were 26.33 % (SD = 2.02) and 0.87 (SD = 0.06), respectively, which were obtained in the seeds submersed in 150 mg·L-1 GA + 30 mg·L-1 6-BA for 8 h under laboratory conditions (Figures 1 and 2). Statistical analyses also showed that GP and GR generally increased with the increase in GA3 + 6-BA concentrations and then decreased with the sustainable increase in these chemical solutions significantly (P < 0.05). From Table 1 shows the interaction between chemical type and duration time, chemical type and chemical concentration affected GP and GR (P < 0.05), whereas the interaction between duration time and chemical concentration had no effect on GP and GR (P > 0.05). Throughout the experiments, the mixed solution of GA3 and 6-BA significantly enhanced the germination of D. giraldii seeds compared to the other treatments; however, this combination did not completely overcome the seed dormancy.
Figure 1. Cumulative germination percentage of the Daphne giraldii seeds germinated at various duration times. 6-BA: 6-benzyladenine; GA3: Gibberellic acid. Treatments: 1) 100 mg·L-1 GA3 + 15 mg·L-1 6-BA, 2) 150 mg·L-1 GA3 + 30 mg·L 6-BA, 3) 200 mg·L-1 GA3 + 45 mg·L-1 6-BA, 4) 250 mg·L-1 GA3 + 60 mg·L 6-BA. The values (mean ± SE, n = 3) marked with the same letter (a-k) in the same frame are not significantly different by LSD test (P ≤ 0.05).
Figure 2. Cumulative germination index of the Daphne giraldii seeds germinated at various duration times. 6-BA: 6-benzyladenine; GA3: Gibberellic acid. Treatments: 1) 100 mg·L-1 GA3 + 15 mg·L-1 6-BA, 2) 150 mg·L-1 GA3 + 30 mg·L-1 6-BA, 3) 200 mg·L-1 GA3 + 45 mg·L-1 6-BA, 4) 250 mg·L-1 GA3 + 60 mg·L-1 6-BA. The values (mean ± SE, n = 3) marked with the same letter (a-h) in the same frame are not significantly different by LSD test (P ≤ 0.05).
Effect of stratification on dormancy of D. giraldii
As shown in Figure 3, stratification obviously increased the total germination of D. giraldii seeds. When the duration of stratification was increased from 30 days to 70 days at 5 °C , both GP and GR significantly increased (P < 0.05), and then significantly decreased (P < 0.05) when stratified for 90 d, showing the same trend as that of 10 °C treatment. The seeds stratified for 30, 50, 70 and 90 d at 5 °C resulted in GP of 12, 22, 52.33 and 44.67 %, respectively. The highest GP and GR were obtain from this treatment; the values were slightly higher than the 10 °C treatment. These results were significantly different from those of the treatment at -5 °C and 0 °C and those obtained from the non-stratified seeds control). At the temperature 0 °C, seeds stratification for 50 d had a low GP but also significantly different from the 30 d duration which did not germinated (P < 0.05), however, there were no significantly difference with the increase of the duration time (70-90 d) (P > 0.05). These results were the same as those of GR. Moreover, the significant variation confirmed in our study can be attributed to the treatment of the seeds stratified for 70 d at 5 °C, 0 °C, and 10 °C treatments, where GP and GR increased, whereas the seeds at -5 °C did not germinated at any stratification period.
Effect of seed burial on dormancy of D. giraldii
The effects of sowing depth and duration of seed burial on GP and GR are summarized in Figure 4 and Table 2. The seeds buried at 20 cm for 190 d had 11.3 % germination, whereas those buried at 100 cm for 170 d 86.5 % germination. D. giraldii seeds buried at 100 cm depth at 100 cm for 170 d exceeded 80 %, in which germination was rapid and the majority of seeds germinated within one week. In the remaining depths, GP decreased for all treatments and the control (obtained from the laboratory at 25 ± 1 °C) did not germinated; these results were the same as those of GR. However, the seeds buried at 20 cm depth had a low GP and GR. Table 2 show that the effects of burial time, burial depth and the interaction between burial time and burial depth significantly affected (P < 0.05) GP and GR of D. giraldii seeds. A significantly interaction effect between burial time and burial depth was apparently caused by a gradual increase over depth of the burial at suitable burial time (Figure 4; Table 2).
Figure 4. GP and GR of Daphne giraldii for various periods. The values (mean ± SE, n = 3) marked with the same letter (a-l) in the same frame are not significantly different by LSD of Tukey's test (P ≤ 0.05).
Table 2. ANOVA of the germination percentage (GP) and germination rate (GR) of Daphne giraldii seeds as result of varying depth and time of burial carried out in 2013 in an open field (split-plot ANOVA).
Less than 1 % of the buried seeds were considered dead in the seed lots exhumed on April 10, 2014. Daily average temperatures at the burial site were between -20 °C and 9 °C from October 2013 to early April 2014 and slightly increased in March. This finding indicates that the dormancy level increased in response to this temperature range outside.
Seed problems related to seed dormancy often limit the use of some species for the production of seedlings.
Different plant species have different seed dormancy classes, which can be divided into morphological, morphophysiological, physiological, physical and combinational groups (Baskin & Baskin, 2004; Gerhard, 2005; Geissler & Gzik, 2010). Xing, Guo, and Wang (2003) reported that the plant Stellera chamaejasme L. which belonged to Thymelaeaceae has the physical and physiological dormancy, and the highest germination was less than 50 % under various pre-sowing treatment. Our experiments show that the seeds of D. giraldii belong to the physiological class according to the Baskin seed dormancy classification system (Baskin & Baskin, 2004). These findings support the conclusion of Wang (Wang et al., 2012) that fresh D. giraldii seeds have this type of dormancy.
Some researchers (Airi, Bhatt, Bhatt, Rawal, & Dhar, 2009; Azad, Rahman, & Matin, 2011) have studied different pre-sowing treatments for seed germination to break seed dormancy and thereby enhance the rate of germination and accelerate the germination process. Seed dormancy may be overcome by chemical treatments, incremental stratification and seed burial (Ooim, Auld, & Whelan 2006; Travlos et al., 2007; Merritt et al., 2007). The results of this study provided several previously unreported insights into the dormancy and germination of D. giraldii seeds. The dormancy level of a seed batch cannot be directly assessed, but can be indirectly measured using germination tests. The findings of the present study showed that the GP and GR of D. giraldii seeds significantly increased (P < 0.05) under different pre-treatments compared with the control. Pre-treatments exhibited that chemical treatment was not very effective in breaking the dormancy (Figures 1 and 2). GA3 and 6-BA can increase and synchronize the seed germination of many plants (Nadjafi, Bannayan, Tabrizi, & Rastgoo, 2006; Chisha, Woodward, & Price, 2007). Tang et al. (2012) and Yang, Ye, Wang, and Yin (2009) reported soaking for 24 h in 250 mg·L-1 GA3 or 50 mg·L-1 6-BA could reach significantly G (40~80 %) for M. dodecandrum and Ardisia crenata Sims. seeds. However, our data showed that the seed germination in the mixed liquor of GA3 and 6-BA solutions at various times shows significant differences with the control (fresh seeds) and higher germination than obtained with other single chemical treatments. The best germination condition of chemically-treated seeds had low GP (less than 30 %) and GR (less than 1.5). The fresh seeds of D. giraldii without pretreatment did not germinate, but the chemical pretreatments slightly improved the germination, which revealed that the embryo exhibited some degree of dormancy that was overcome by chemical treatments partially. Chemical treatments were not completely effective in breaking the dormancy, which could be attributed to the basic differences in the mechanisms that impose dormancy with other species. Baskin and Baskin (2001) indicated that the dormancy of Plumbaginaceae and Juncaginaceae is physiological in nature and can be broken by cold stratification. Stratification is widely used to break physiological dormancy and enhance the germination of seeds in numerous species (Baker, Steadman, Plummer, & Dixon, 2005; Farshad, Hojat, & Mahmood, 2012). In the present study, the seeds of D. giraldii stratified for a period of time responded favorably to chemical treatments. The seeds stratified for 70 d at 5 °C increased GP (52.33 %) and GR (1.19), whereas the lowest germination (0 %) was found in the stratification treatment at -5 °C for 90 d, and 0 °C for 30 d; similar result were reported by Sechenbater and Am (2002). GA3 and 6-BA promotes seed germination and stratification breaks dormancy at a higher degree, thus D. giraldii seeds are classified as having physiological dormancy (Baskin & Baskin, 2004; Nikolaeva, 1977). Recent physiological studies have shown that physiological dormancy includes an embryo and coat component, and their sum and interaction determine the degree of whole seed physiological dormancy (Chen, Kuo, & Chien, 2008). In our study, cold stratification had better effects than chemical treatment on relieving dormancy and the higher GP had been attained in pretreatments of D. giraldii seeds by prolonged cold stratification at 5 °C for 70 d, which was better than 10 °C treatment, this can be explained by this condition where it may be beneficial for softening the seed coat in moist sand and eliminating germination inhibitors under a suitable temperature (5 °C) (Packa, Kwiatkowski, & Graban, 2014).
Our results showed that the physiological dormancy of D. giraldii seeds was effectively alleviated during burial. The Hexi university is located in the temperate desert and the edge of Qilian Mountains, the soil composition in this area were clay loam, silt loam soil and sandy loam soil 0~150 cm below the surface (Li, Qi, Zhao, & Zhang, 1999), burial treatments alleviated seed dormancy and thus increased the GP and GR of D. giraldii in this area. The seeds buried on October 21, 2013 at 100 cm depth and recovered on April 10, 2014 (seeds remain in soil for 170 d) showed the highest germination success with GP of 86.5 % and GR of 10.11. This treatment was also the most effective in alleviating seed dormancy, demonstrating that the period between October 21, 2013 and April 10, 2014 is likely to be a predominant period and appropriate burial time (170 d) for releasing seed dormancy of D. giraldii seeds in this area. Among those burial treatment, increasing the burial depth up to 100 cm significantly enhanced GP and GR, and the seeds buried at 100 cm depth had higher GP and GR than those buried at soil depths of 20, 40, 60, 80 and 120 cm. GP and GR slightly decreased when soil depths continuously increased (120 cm), This is perhaps as a result of the low oxygen level at greater soil depths, a very low oxygen levels at a certain soil depth may lead to induction of secondary dormancy (Malik & Vanden, 1988; Poinar & Columbus, 1992). The buried seeds of D. giraldii could be in a hypoxic environment, and seed germination may be affected by the variation in soil oxygen availability associated with different burial depths (Yan, Liu, Li, & Ma, 2007). However, some study work is needed to explore the oxygen levels in various burial depth of this area further. The results of our study differ from the findings reported by other authors in recent years. In a China study the germination of northwest shrub seeds A. ordosica was enhanced by burial at 5 cm depth for six months (Liu et al., 2013), whereas another Turkey study reveals that the greater depths (> 20 cm) would possibly induce seeds into secondary dormancy (Mennan, 2003). The above discrepancies could result from genetic variations in the studied populations, environment conditions and soil composition and treatment applied before germination. During winter, the surface of the land in the experimental area is frozen because the temperature falls to -20 °C outside (Li, Gao, Wang, & Wang, 2013). In early spring, the temperature increases from 5 °C to 9 °C, and the soil becomes wet because of melting ice and snow. Thus, the cold winter season is likely to be a predominant period for releasing seed dormancy in D. giraldii, and the 170 d burial period effectively affected and enhanced germination. The physiological nature of the embryo dormancy in D. giraldii is not yet clearly elucidated but a possible explanation is given by Walker (1971), who showed that dormancy is controlled to some extent by some inhibitors in the seed. These inhibitors are possibly formed at an early stage in the development of the seed to prevent the germination process. In this study, the response may be due to the better degradation of germination inhibitors in the embryo over the 170 d burial time in this area under appropriate depth of soil. Thus, we recommend using a more appropriate burial treatment to break dormancy in future germination studies on D. giraldii. This information may be useful to cultivate and conserve other shrubs grown in the Qilian Mountains in China and elucidate their survival under similar extreme environments.
Conclusions
From this research, it can be concluded that the very poor natural regeneration of D. giraldii is mostly attributed to physiological dormancy. This study indicated that seed stratification at 5 °C for 70 days appeared to be a more effective method to break seed dormancy of D. giraldii than chemical treatment. However, seeds were buried in sandy loam at 100 cm depth for 170 days to offer the most convenient and effective dormancy breaking method for D. giraldii. These findings make a significant contribution to the conservation efforts for this endemic species grown in Northwestern China.
