2.1. Plant and irradiation

The sweet sorghum seeds, Sorghum bicolor (L.) Moench “KFJT-CK,” were provided by the Institute of Modern Physics of the Chinese Academy of Sciences. Dry seeds of equal size without mold or lesions were selected. The carbon ions were generated by the Heavy Ion Research Facility in Lanzhou (HIRFL) facility, Institute of Modern Physics, CAS (IMP). The energy of the carbon ions was 100 MeV/u, and the dose rate was approximately 20 Gy/min. The dry seeds were irradiated by the carbon ions with the doses of 0, 20, 40, 60, and 80 Gy. The early maturity mutants (KF1210-3 and KF1210-4) were screened with the irradiation dose of 80 Gy and bred for further experiments.

2.2 Soil sampling and assays

After the establishment of the plots and before sowing the sweet sorghum (i.e., initial soil condition before the implementation of the treatments), composite soil samples were collected from each experimental plot at 0–20 cm depths. Soil sampling was also undertaken after the harvest of sweet sorghum in each cropping year. The collected samples were processed (air-dried, ground to pass through a 2 mm sieve, and mixed thoroughly) before being subjected to the following soil analyses: salinity, pH, sodium (Na⁺), and potassium (K⁺) via flame photometry. All methods were according to those described by the U.S. Salinity Laboratory Staff. Sodium adsorption ratio (SAR) was calculated via concentrations of Na⁺ from the saturation paste extract. The experimental results are shown in Table 1.

2.3 Treatments and experimental design

The experiment was established in the industrial park of Wuwei in China (approximately 20 hm²), which exhibits great variability in soil salinity. The sweet sorghum seeds that had been irradiated by carbon ions were sown in the field on April 26, 2014. The sweet sorghum was harvested on October 2, 2014. The soil salinity was within the range of 3.2 dSm⁻¹-12.6 dSm⁻¹, and the soil pH was approximately 8.5. Each treatment was replicated three times according to a randomized block design. A plot size of 240 m² was adopted. The space between rows was 50 cm, and plants were spaced 20 cm apart within a row. This spacing conforms to typical sweet sorghum protection and irrigation practices [14]. In the experimental field, the two saline soil blocks (80 m x 34 m; 2720 m²) were selected on the basis of their salinity level (4.6 dSm⁻¹ and 11.9 dSm⁻¹). The two salinity blocks (part 1 and part 2) were separated by a wide alley (50 m) to maintain the integrity of each salinity level.

The three sweet sorghum cultivars (KF1210-3, KF1210-4, and KFJT-CK) were planted in rows (20 cm) by hand in the block design to achieve an approximate density of 500,000 seeds ha⁻¹. Before sorghum planting, 260 kg N ha⁻¹ as ammonium sulphate ((NH₄)₂SO₄), 100 kg P ha⁻¹ as super phosphate (Ca(H₂PO₄)₂), and 195 kg K ha⁻¹ as potassium sulphate (K₂SO₄) were applied and incorporated into the soil of all experimental plots. After planting, all experimental plots were irrigated with 30 mm of water for the emergence of the sweet sorghum. The salinity of water used for irrigation was lower than 1.0 dSm⁻¹ to avoid additional adverse effects, especially on the sweet sorghum biomass. The growth-stage irrigation began in early July and was sustained until harvest. During this period, the entire area was irrigated four times with 120 mm of water. The barnyard grass was hand-removed. Other agricultural practices were carried out according to the recommended production practices for sorghum fields in China.

2.4 Measurement of agronomic characters

The fresh biomass of the sweet sorghum was determined by hand-harvesting at the hard dough stage of each sorghum cultivar, which was approximately 35 days after blossom. The sweet sorghum provides the greatest biomass and total sugar yield at this stage. Ten randomly selected plants were chopped into 1 cm long pieces, and 4 kg of chopped biomass samples was used for determination of dry biomass yields. The Brix degree of the sweet sorghum juice was measured with an automatic lab refractometer (Model ATR-ST, Topac, MA, USA).

2.5 Physiological assays

The chlorophyll content was determined from the same leaf samples used for the reflectance measurement. The method of measurement was conducted according to Lichtenthaler [15]. The proline content was estimated using the acid-ninhydrin method [16]. Ten stems and ten roots from the five marked plants of each plot were collected for determination of the Na⁺ and K⁺ ratio at the physiological maturity stage. The samples were dried at 65 °C for 72 h and were then ground in a Wiley mill to pass through a 1 mm screen. The sodium ions (Na⁺) and potassium ions (K⁺) in the roots and stems were determined via an atomic absorption spectrophotometer (AA, Model M6, Thermo Elemental, MA, USA) [17]. In addition, the K⁺/Na⁺ ratio was calculated and used for further data analysis.

2.6 Statistical analysis

Statistical analysis was performed using the Statistical Analysis System (SAS) computer program. The means were compared according to the Duncan multiple range test.

3.1. The emergence and yields of the sweet sorghum

In particular, the plant density of the sweet sorghum in the soil salinity of 11.9 dSm⁻¹ was 37% lower (43,614 plants ha⁻¹) than that in the soil salinity of 4.6 dSm⁻¹ (69,275 plants ha⁻¹). Averaged across the two soil salinity levels, the KFJT-CK sweet sorghum cultivar (47.753 plants ha⁻¹) was seriously affected by the soil salinity. The KF1210-3 and KF1210-4 sweet sorghum showed greater levels of emergence (56,467 plants ha⁻¹ and 53, 564 plants ha⁻¹, respectively). The effects of soil salinity and cultivar on fresh biomass, dry biomass, and the Brix degree of the sweet sorghum juice are shown in Table 2. The fresh biomass of the KFJT-CK, KF1210-3, and KF1210-4 sweet sorghum cultivars ranged from 43.33 t ha⁻¹ to 66.52 t ha⁻¹, 41.85 t ha⁻¹ to 65.49 t ha⁻¹, and 37.22 t ha⁻¹ to 55.64 t ha⁻¹ in the different salinity conditions (11.9 dSm⁻¹ to 4.6 dSm⁻¹), respectively. The dry biomass of the KFJT-CK, KF1210-3, and KF1210-4 sweet sorghum cultivars ranged from 14.89 t ha⁻¹ to 26.36 t ha⁻¹, 14.32 t ha⁻¹ to 25.21 t ha⁻¹, and 13.08 t ha⁻¹ to 18.89 t ha⁻¹, respectively. By comparing the three sweet sorghum cultivars under conditions of increasing soil salinity, it was found that the fresh biomass of sweet sorghum was reduced with increasing soil salinity. However, the results did not show significant differences in dry biomass between salinity treatments. In addition, the Brix degree values of the KFJT-CK, KF1210-3, and KF1210-4 sweet sorghum cultivars ranged from 14.68% to 15.08%, 20.37% to 22.50%, and 21.09% to 22.49% with the increasing salinity. Netondo et al. found that when the external NaCl concentration increased during the sensitive vegetative phases, both the sorghum leaf growth and leaf area decreased. This led to lower biomass production [18].

3.2. Physiological parameters

In plants, chlorophyll a can capture photons that are scarcely absorbed by chlorophyll b in photosynthetic light-harvesting complexes. In most cases, the increasing soil salinity caused a decrease in the total plant chlorophyll. In this experiment, the chlorophyll content index (CCI) of sweet sorghum in the bloom growth stages was affected by the soil salinity and sorghum cultivar type. The increase in soil salinity decreased the sweet sorghum CCI from 8% to 15% (Fig. 1). This result may be related to the mechanism of salt tolerance in sweet sorghum, and it may aid in screening for tolerant sweet sorghum cultivars. Under greenhouse conditions, Netondo et al. found that the sorghum Chlorophyll a and Chlorophyll b are reduced from 58% to 70%, while the net CO₂ uptake is almost completely inhibited when the external NaCl concentration increased to 200 mM [19].

Fig. 1.Full-screen View

The chlorophyll content index (CCI) of sweet sorghum was affected by the type of sweet sorghum cultivar and soil salinity of (a) 4.6 dSm⁻¹ and (b) 11.9 dSm⁻¹.

Proline is a solute that helps regulate cellular osmolarity during stress in plants, and it is an effective cryoprotectant against natural stresses [20, 21]. Under salt stress conditions, tolerant sweet sorghum cultivars can accumulate proline to prevent changes in plasma membrane permeability as well as protect the integrity and stability of the plasma membrane proteins. At the same time, the sweet sorghum can maintain high osmotic pressure stress to absorbed moisture under salt stress. In Fig. 2, the proline content exhibits little change between the sorghum cultivars in the soil salinity of 4.6 dSm⁻¹. In contrast, proline content of KF1210-3 and KFJ1210-4 are higher than KFJT-CK in the soil salinity of 11.9 dSm⁻¹. The proline content of KFJT-1210-3 was increased by 36.94% compared to KFJT-CK. The result indicates that KF1210-3 has the strongest resistance to the salt stress. Carbon ion irradiation may change a key gene of the proline metabolic pathway of sweet sorghum. Blum and Ebercon also suggested that proline has an active role in recovery metabolism [22]. Liu et al. reported that the levels of proline in transgenic rice increased over threefold compared with the levels of proline in WT rice after 4 °C cold treatment for 24 h [23].

Fig. 2.Full-screen View

Changes in the content of proline in KFJT-1210-3, KFJT-1210-4, and KFJT-CK with increasing salinity. The means with the same letter within each cultivar are not significantly different according to the Fisher’s protected LSD test at P = 0.05.

3.3. Effect of soil salinity on K⁺/Na⁺ ratio and sugar accumulations of sweet sorghum

In the salt-affected soil, Na⁺ is a small molecule that is easily absorbed into root tissues and transported throughout plant organs, leading to toxic ion damage, osmotic stress, and nutritional imbalance. In halophyte species, there are many salt-defense mechanisms, including ion homeostasis, osmoregulation, antioxidant, and hormonal regulation. An increase in stem or root K⁺/Na⁺ ratio has been suggested as a possible ion homeostasis mechanism of the sweet sorghum salt stress tolerance [24]. Thus, the K⁺/Na⁺ ratio can be utilized to identify salt-tolerant or salt-sensitive varieties [25, 26]. In order to maintain the balance of K⁺/Na⁺ in the cytoplasm under salt stress, the salt-tolerant sweet sorghum varieties could reach ion homeostasis via inhibiting Na⁺ accumulation and accelerating K⁺ accumulation in the cytoplasm. Generally, the K⁺/Na⁺ ratio in the sweet sorghum stems and roots was affected by the sweet sorghum cultivar type and soil salinity. The results are shown in Fig. 3. The Na⁺ increased with the increase in the soil salinity, while the K⁺ was not significantly related to the soil salinity. The Na⁺ in the sweet sorghum KF1210-3 root was highest, and the opposite result was obtained in the sweet sorghum KF1210-3 stem. Meanwhile, the K⁺/Na⁺ ratio (Fig. 4) in the sweet sorghum stems decreased with the increase in the soil salinity. These results suggest that the sweet sorghum under the salt stress can inhibit the motion of Na⁺ from root to stem in order to maintain ion homeostasis in cytoplasm.

Fig. 3.Full-screen View

(A) The sodium ions (Na+) of sweet sorghum (a) roots and (b) stems of three sorghum cultivars grown in two soil salinity conditions; (B) The potassium ions (K+) of sweet sorghum (a) root and (b) stem of three sorghum cultivars grown in two soil salinity conditions.

Fig. 4.Full-screen View

The K⁺/Na⁺ ratio of (a) roots and (b) stems of the three sweet sorghum cultivars grown in two soil salinity conditions.

On the other hand, the Na⁺ was positively related to total soluble sugars. The increase of soluble sugars in the sweet sorghum stems may play a key role in the osmotic adjustment to prevent water loss in the plant cells during salt stress [27, 28]. The Brix degree content of KF1210-3 and KF1210-4 significantly increased with the increase of soil salinity. Silva et al. reported that in sorghum under salt stress, salt-tolerant cultivars could be enhanced in the soluble carbohydrate content. They suggested that the accumulation of soluble carbohydrates were significantly related to salt tolerance due to leaf osmotic adjustment under the salt stress. Juan et al. also reported that the sucrose content is an indicator of the salt tolerance in tomato [29].