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INTRODUCTION
Compared to other conventional crops, sweet sorghum (Sorghum bicolor [L.] Moench) can survive in lower quantity of water and fertilizer. It is a potential feedstock crop in biomass energy development. Sweet sorghum is much more resistant to saline soils than corn (Zea mays L.) or sugarcane (Saccharum officinarum L.), which are currently the main bioenergy resources in the world (Almodares et al., 2011). Sweet sorghum crops have a great potential for manufacturing syrups for sweetening food and beverages, carbohydrates, and most importantly, as a raw material for fuel alcohol production worldwide (Ratanavathi et al., 2004). In China, people can take advantage of broad saline lands by planting salt-tolerant sweet sorghum varieties. Although it has been found that a number of sweet sorghum varieties can survive under salinity soil condition, however, their seed germination and seedling establishment are still difficult on marginal saline lands.
Currently, more than 900 million hectares or about 20% of the total agricultural land are affected by salinity around the world. Salinity is becoming an increasingly serious problem limiting crop production worldwide (Munns and Tester, 2008). Salinity can affect crop growth from germination stage, the very beginning of the life cycle of a crop plant. Healthy seed germination plays an important role in the growth cycle of plants, and determines the establishment of seedlings and subsequent crop production (Bahrani and Pourreza, 2012). High salinity conditions can result in difficult seed germination and delays in the germination time (Nyagah and Musyimi, 2009). The inhibition of salinity on seed germination was mainly due to water deficit and/or the toxic influence of ions, such as absorbing excessive Na+ and Cl- ions (Murillo et al., 2002).
The water uptake is the first stage preparing for seed germination. Under adequate supply of oxygen and optimal temperature conditions, the most important factor in seed germination is water status. Viable seeds have the ability to break dormancy and begin germination after absorbing enough water. However, water quality varies greatly caused by degrading environments and salinization has an important effect on inhibiting germination and subsequent root elongation (Saberali and Moradi, 2017). It was reported that water uptake of tomato plants declined with increasing salinity, causing significant reductions in morphological and/or physiological parameters, stomatal density and water conductance (Romero et al., 2001).
Plant hormones are active members of plant regulation, and are involved in the induction of plant stress responses (Pedranzani et al., 2003). They make plants adapt to serious abiotic stress conditions, and help crops to improve their tolerance, capacity to adverse environments (Srivastava and Srivastava, 2007). Gibberellic acid (GA3) is an important plant hormone, which plays a vital role in regulating the signal pathways, seed germination and plant growth (Cavusoglu and Sulusoglu, 2015). It has been shown that GA3 is related to salinity tolerance of Arabidopsis (Arabidopsis thaliana L.) (Sun, 2008).
There is abundant evidence showed that GA3 has a positive effect on water uptake and germination of crops under normal conditions, but little research is in the literature on sweet sorghum grown in saline soils. Furthermore, the related mechanism of GA3 in regulating germination is still not well documented. Therefore, the objectives of this research were to explore the effects of GA3 amendment on water uptake and germination of sweet sorghum at different salinity levels; and screen the optimal concentrations of GA3 to sweet sorghum germination under saline conditions.
MATERIALS AND METHODS
A controlled study was conducted in Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education of China, Yangzhou University (32.30º N, 119.25º E), Jiangsu Province, China. The seeds of sweet sorghum ‘Chuntian 1’, kindly provided by Beijing Sangliang Technological Development Center, were used. The variety is relatively salt-tolerant and being popularly grown in China.
Material and cultivation
The study was arranged in two-factorial randomized complete block design with three replicates. The two factors were NaCl (0, 50, and 100 mM water solutions) and gibberellic acid (GA3; 0, 144, 288, and 576 μM water solutions). For each replicate of each treatment, 50 uniform and healthy seeds were selected and surface-sterilized using 1% sodium hypochlorite solution for 10 min, and then rinsed thoroughly six times with deionized water and dried by air, and fresh weight was considered as initial weight. After that, all the seeds were cultivated in Petri dishes with treatment solutions. The diameter of the Petri dish was 9 cm. A double layer of filter paper was placed in each Petri dish, and 7 mL of different treatment solutions of NaCl and GA3 were infused. Then the Petri dishes were covered with lids and placed in a germinator (Model ZLC-100, Hangzhou Shuolian Instrument Co., Ltd., Hangzhou, Zhejiang, China) with a natural light 12:12 h diurnal cycle, constant temperature 25 °C and humidity 60%. In order to maintain the treatment levels, each Petri dish was carefully injected with 5.0 mL treatment solutions every 8 h to replenish evaporation and solution absorbed by seeds. The filter paper was changed every 48 h during the testing period.
Observations and measurements
The seeds in each Petri dish were weighed before soaking and during seed water uptake at 8, 16, 24, 32, 40, 48, 56 and 64 h after the beginning of water imbibition. In order to measure seed cumulative water uptake and water uptake rate, seeds were carefully removed, drained, blotted with absorbent paper, weighed, and, returned to Petri dishes quickly. Germination parameters, including cumulative water uptake, cumulative germination, and germination index at each measurement time were calculated as the following:
where X 0 is the initial weight of the 50 sweet sorghum seeds (g) and Xt is the weight at t (h).
where S t is the number of seeds germinated at t (h) and S 0 is the initial number of seeds (50) subjected to the germination test at time t = 0 h.
where G i is germination index at time t i (h), count i is the number of seeds germinated at time ti, count i-1 is the number of seeds germinated at time ti-1, S 0 is the number of seeds subjected to the germination test, and t i is observation time.
Seeds were considered to be germinated when radicle length reached approximately 0.2 mm. Both radicle and germ lengths were precisely measured using a vernier caliper after 32 h. The test was terminated after 64 h in the germinator.
Statistical analysis
The experiment was designed as a factorial design with two experimental factors (three salinity levels, and four hormones gradients) arranged in a completely randomized design with three replicates. The data of each variable were then subjected to ANOVA with the statistical package of DPS 7.05 for Windows (Tang and Feng, 1997) according to this design. When “F” values were significant, means were separated by the least significant difference test (LSD, P ≤ 0.05).
RESULTS
Cumulative water uptake
There was nonsignificant effects of salt, GA3 or Salt × GA3 interaction on cumulative water uptake at the first 32 h except Salt × GA3 interaction at 16 h (Table 1). The effect of salt on cumulative water uptake was nonsignificant at any time point, but the cumulative water uptake was significantly affected by GA3 and Salt × GA3 at 40 and 48 h, 16 and 48 h, respectively (Table 1). Some of the pairwise comparisons were significantly different from each other as affected by Salt × GA3 at 16-48 h. Cumulative water uptake was inhibited at whole germination period at high GA3 or high salt. Compared with control, the cumulative water uptake was increased by about 8%-12% at 50 mM salinity level during 8-56 h. However, when salinity was increased to 100 mM NaCl, the cumulative water uptake was decreased by about 14%-16% during the period of 8-48 h. Statistical analysis indicated that the inhibition of seeds cumulative water uptake caused by salinity could be alleviated by application of GA3. With increased concentration of GA3, the cumulative water uptake was gradually increased at low and middle level but decreased at high level of GA3 at 100 mM NaCl. In general, water uptake was promoted by 288 μM GA3 but then decreased at 576 μM GA3 under each salinity level during whole germination period. Salinity and GA3 treatments showed the most prominent effect during the time period of 32-48 h, the highest cumulative water uptake was recorded at 50 mM NaCl and 288 μM GA3 (Table 2).
Cumulative germination
The ANOVA showed that cumulative germination of sweet sorghum seeds were significantly influenced by salinity during the germination period, but by GA3 only during 32-48 h (P ≤ 0.05 and P ≤ 0.01). However, Salt × GA3 had nonsignificant effects on cumulative germination in any growth period (Table 1). Compared with control, 50 mM NaCl significantly increased the cumulative germination by 23% at 8 h and 17% at 16 h, but then declined with 54% and 42.2%, respectively, when salinity concentration was increased to 100 mM NaCl. Compared to non-GA3, cumulative germination was significantly increased at the levels of 144 and 288 μM GA3 during 32-48 h, but decreased at 576 μM GA3 at all salinity levels. For example, the cumulative germination declined by 7.5% from 288 to 576 μM GA3 at 50 mM NaCl at 48 h (Table 3). As a whole, during the germination period of 32-56 h, the highest cumulative germination was presented by applying 288 μM GA3 regardless of the salinity levels.
Table 1 Significance levels of salt, gibberellic acid (GA3) and Salt × GA3 interaction on different germination parameters for sweet sorghum seeds at different measurement times.
*, **, ***Significant at the 0.05, 0.01, 0.001 probability levels, respectively. ns: Nonsignificant; -: no measurements at that time.
Germination index
According to the ANOVA, salt stress had a significant effect on germination index, but GA3 had not except at 48 h. The Salt × GA3 interaction did not significantly affect germination index at any sampling time (Table 1). Compared with control, germination index was slightly decreased at 50 mM NaCl but it was significantly inhibited at 100 mM NaCl (Figure 1). At the level of 0 and 100 mM NaCl, germination index was improved by 144 and 288 μM GA3, but it was suppressed at 576 μM GA3 and 50 mM NaCl. At each salinity level, the highest germination index was recorded at 0 mM NaCl by 144 μM GA3, 50 mM NaCl by 0 μM GA3, and 100 mM NaCl by 288 μM GA3 (Figure 1).
Table 3 Cumulative germination of sweet sorghum seeds as influenced by gibberellic acid (GA3) amendments at different salinity levels.
Means in the same column and followed by the same letter indicate nonsignificant difference (P ≥ 0.05).
Radicle length
Salinity, GA3 and Salt × GA3 significantly affected radicle length mainly after 48 h water absorption (P ≤ 0.001) (Table 1). Radicle length was improved at the low salinity level but greatly inhibited under high salt stress. Compared with 0 mM NaCl, radicle length was increased by 5%-6% at 50 mM NaCl during 48-64 h. With rising salinity to 100 mM NaCl, radicle length was significantly decreased by 51%-18% during 8-64 h. The reduction in radicle length caused by salinity was significantly alleviated by external application of 144 and 288 μM GA3. Under 100 mM NaCl, radicle length was increased by 26%-22% when applied 288 μM GA3 during 40-64 h, but it was prominently inhibited by 576 μM GA3 under all salt treatments. Overall, the radical length was prominently enhanced by application of 288 μM GA3 during all growth period from 32 to 64 h under each salinity treatment except at 32 h with 0 mM NaCl (Table 4).
Germ lengthTable 5
The effects of salt, GA3 and Salt × GA3 were significant on the germ length at 32, 40, and 48 h after water uptake initiation (P ≤ 0.001) (Table 1). On the average, there was nonsignificant difference in germ length between 0 and 50 mM NaCl treatments. However, compared to 0 mM NaCl, germ length was decreased by 37%-12% at 100 mM NaCl during 32-56 h.
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The ratio of radicle length to germ length
The effects of salt, GA3 and Salt × GA3 on the ratio of radicle length to germ length mainly came up after 40 h of germination (P ≤ 0.001) (Table 1). The concentration of 144 μM GA3 significantly improved radicle length/germ length at 0 mM NaCl at 32, 48 and 64 h, and at 50 mM NaCl during all germination period except at 48 h. However, under 100 mM NaCl, radicle length/germ length was promoted by 288 μM GA3 at 32 and 40 h, and by 576 μM GA3 at 56 and 64 h (Table 6).
Table 5 Germ length of sweet sorghum seeds at different times as influenced by gibberellic acid (GA3) amendments at different salinity levels.
Means in the same column and followed by the same letter indicate nonsignificant difference (P ≥ 0.05).
DISCUSSION
Salinity inhibits water uptake, delays seed germination, slows down growth rate, changes metabolic and reduces biomass production (Munns, 2002). In most cases, salinity does harm plant survival by disturbing different plant mechanisms (Tavakkoli et al., 2010). Salinity at higher levels usually causes both hypertonic and hyperosmotic stresses and can lead to plant death. These effects may cause membrane damage, nutrient imbalance, altered levels of growth regulators, enzymatic inhibition and metabolic dysfunction (Sudhir and Murthy, 2004), changes in C and N metabolism (Kim et al., 2004), and decreased photosynthesis rate, which ultimately leads to plant death (Mahajan and Tuteja, 2005; Hasanuzzaman et al., 2012). Conventional crops, such as wheat and rice, are sensitive to high salinity conditions and their biomass production and yield gain are impeded sharply at high salinity levels (Bahrani and Haghjoo, 2011).
However, as for sweet sorghum, it has been proved to be more tolerant to salt stress as compared with conventional crops aforementioned (Ratanavathi et al., 2004), but the poor germination under severe salt stress is still a crucial problem to limit sweet sorghum production. An increasing evidence showed that the growth of sorghum is seriously restrained at 250 mM rather than at 125 mM NaCl (Ibrahim, 2004). In the present study, cumulative water uptake, cumulative germination and germination index were all exhibited with similar tendencies at 100 mM NaCl. These results are consistent with Yang and Li (2014). These damage to seed germination and seedling growth is closely related to Na+ accumulation. Excessive accumulation of Na+ can cause a range of osmotic and metabolic problems for plants (Hoai et al., 2003). Most toxic effects of NaCl can be attributed to Na+ toxicity that can result in the dormancy of seeds and delay germination. It is known that toxic effects of Na+ are largely due to its ability to compete with K+ for binding sites, essential for cellular function (Yildirim et al., 2009). As a major plant macronutrient, K plays important roles on stomatal behavior, osmoregulation, enzyme activity, cell expansion, neutralization of non-diffusible negatively charged ions, and membrane polarization (Qin et al., 2010). On the other hand, Na and Cl ions can enter into the cells and have direct toxic effects on cell membranes, as well as on metabolic activities in the cytosol (Cha-Um and Kirdmanee, 2010).
On the contrary, slight salinity improved germination parameters at the level of 50 mM NaCl. Similar results were found by other scientists (Nimir et al., 2017). There is evidence that low salinity sometimes stimulates photosynthesis of Bruguiera parviflora. Parida et al. (2004) observed that the rate of photosynthesis increased at low salinity while decreased at high salinity. But the related mechanism is still not clear.
The capacity of crops tolerant to stress can be improved by a number of ways, including selection and breeding, genetic modifications, and use of osmoprotectants and growth regulating substances (Parida et al., 2004). In this regard, attention has come to be focused on the use of plant growth regulators, such as GA3, kinetin, and salicylic acid, which are known to regulate plant responses to adverse external environments and to regulate the expression of a number of stress-induced genes. At high salinity levels, the germination of sweet sorghum is deteriorated. The osmotic regulation, together with the toxic effects of Na+ and Cl- ions, reduces water uptake and causes an imbalance of essential nutrients during seed germination (Willenborg et al., 2004). In the present study, we observed that the cumulative water uptake, cumulative germination, germination index and the length of radicle and germ of sweet sorghum seeds were significant improved by applying 144 and 288 μM GA3. Similar results were found in cotton (Gossypium barbadense L.) and castor (Ricinus communis L.) seeds when GA3 was amended at appropriate concentrations (Zhou et al., 2014). Also with wheat seeds, the greatest improvement in seed germination was achieved when seeds were presoaked in 50 mg GA3 L-1 (Parashar and Varma, 1988). As for sweet sorghum, the optimum GA3 concentration to promote seed germination is 288 μM GA3 in this study. At this level, most of the parameters were improved under each salinity levels during different germination periods (Tables 2-6).
The probable mechanism is that GA3 can break seed dormancy, stimulate seed embryos, thereby promote plant metabolic reactions, repair the integrity of damaged cell and improve seed viability. Nimir et al. (2017) reported that GA3 caused a reduction in Na+ content and partly decreased the content of other ions. These results agreed with those of Kaya et al. (2010), who reported that stressed maize plants significantly accumulated less Na+ upon application of GA3. In another study, application of GA3 counteracted the adverse effects of NaCl salinity on relative water content, electrolyte leakage, and chlorophyll content (Ahmad et al., 2011).
According to previous studies, external seed treatment with GA3 could be a possible method of reversing the effects of salt stress (Tuna et al., 2008). However, when GA3 level was increased to 576 μM, most germination parameters were suppressed as shown in the present study (Tables 2-6). This results indicated that application of low levels of GA3 can regulate plant growth and have positive effects, but high levels of GA3 may have opposite effects. This result is similar to Baskin et al. (1998), who reported that high level of GA3 reduced the ratio of radicle length to germ length, but the suitable GA3 concentration improved the growth rate of the germ relative to the radicle. The application of 288 μM GA3 can relieve the harmful effects of salinity on water uptake and germination of sweet sorghum. Nevertheless, these results need to be further confirmed in field environment due to the difference in soil environment and NaCl water solution.
CONCLUSION
Sweet sorghum can be tolerant to mild salinity stress, it can be recognized as one of the excellent candidate crops used to exploit the area of coastal shoaly land. Low concentrations of salinity and gibberellic acid (GA3) can enhance water absorption and germination of sweet sorghum, but high levels of these treatments can bring the opposite effects. The 288 μM GA3 is the optimum concentration which can be applied to promote seed germination of sweet sorghum under salinity stress conditions.
