Abstract
Sweet sorghum (Sorghum bicolor L. Moench) is one of the most important crops for bioethanol production and the provision of antioxidant compounds for human health. In this study, we investigated the 8 agronomic traits of 153 sweet sorghum germplasms, which demonstrated a variety of phenotypes. In particular, nine sweet sorghum germplasms exhibited a sugar content exceeding 20 Brix. Based on agronomic characteristics, we selected eight elite sweet sorghum germplasms that showed high-performance agronomic and growth characteristics such as tall height, large panicle size with short height, high sugar content, and seed-specific characteristics. The selected germplasms also showed significant differential amount of 3 antioxidant compounds of 3-deoxyanthocyanins, flavonoids, and tannins. SS113 contained the highest levels of total 3-deoxyanthocyanins with apigeninidin contents 3–9 fold higher than that of other germplasms, while SS129 had a white seed coat with the highest recorded total flavonoid level (7.52 mg/g) but no detectable 3-deoxyanthocyanins compounds. The characterization of the traits and compounds will be useful for basic research into the selection of suitable cultivars in the breeding of sweet sorghum.
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Key words: Sweet sorghum, Antioxidants, Agronomic trait, Sugar content
INTRODUCTION
Sorghum, a popular cereal grass worldwide, is used as foodstuff, animal feed, and fibers in more than 30 countries. In Asia and Africa, it is mostly used directly as foodstuff, whereas it is used in America as animal feed and a resource for industrial products (
Dykes et al. 2009;
Ryu et al. 2016). Sorghum is classified into three types according to the purpose of its application: grain sorghum, which can be short with large ear heads and relatively easy to harvest with a combine; forage sorghum, a tall, thick-stemmed silage crop; and sweet sorghum, a juicy, sugary sorghum that is taller than grain sorghum and has a higher biomass. Sweet sorghum is a natural variant of common grain sorghum with a higher sugar content. Stalks of sweet sorghum are thicker and fleshier than those of grain sorghum, though its seed productivity is relatively low (
Codesido et al. 2013). It is increasingly used as a source of bioethanol because of its various advantages such as a higher reducing sugar content compared with sugarcane juice, which prevents crystallization and results in a near complete fermentation efficiency in bioethanol production (
Guigou et al. 2011;
Mathur et al. 2017).
Sorghum is a C
4 photosynthesis crop that adapts to various environmental conditions by exhibiting different phenotypes (
Mathur et al. 2017). The characterization of agronomic and morphological traits of different sorghum genotypes is important in selecting high nutritional value and productivity (
Santos et al. 2013;
Jahansouz et al. 2014), so it is necessary to investigate these traits to identify the most suitable variety for different applications.
Sorghum plants are a rich source of various phytochemicals such as phenolic acids, anthocyanins, and tannins (
Awika and Rooney 2004). They contain a unique type of anthocyanin, 3-deoxyanthocyanin, which has an increased stability at high pH levels compared with common anthocyanins (
Awika et al. 2004), and is present at higher levels in sorghum with a black pericarp than red sorghum (
Dykes et al. 2005). Apigeninidin (yellow) and luteolinidin (orange) are two types of sorghum 3-deoxyanthocyanidins (
Dykes and Rooney 2006). These compounds showed antioxidant activity as measured by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and α, α-diphenyl-β-picrylhydrazyl free radical scavenging assays (
Awika et al. 2003;
Dykes et al. 2005). Tannins are widely found in grains such as sorghum with a pigmented testa layer, some finger millets, and barley, but not in major cereal crops including rice, wheat, and maize (
Dykes and Rooney 2007). They are involved in various biochemical functions including protection against pathogenic attack from bacteria and consumption by herbivores. Moreover, diets rich in tannin-containing foodstuffs are reported to have beneficial effects include immunomodulatory and anticancer activity, antioxidant and radical scavenging functions, antithrombotic effects, and UV-protective functions (
Dixon et al. 2005;
Sharma et al. 2007;
Wu et al. 2012). However, although tannins support human health through their antioxidant activity, some negative impacts including antinutritional effects and low feed efficiency have been documented (
Chung et al. 1998;
Awika and Rooney 2004).
In this study, we investigated the agronomic and morphological characteristics of 153 sweet sorghum germplasms with the aim of selecting elite sweet sorghum germplasms for growth in a temperate climate. These germplasms were shown to contain a range of levels of phytochemical compounds including 3-deoxyanthocyanidins, total flavonoids, and tannins.
MATERIALS AND METHODS
Plant materials
To investigate agronomics traits, a collection of 153 sweet sorghum germplasms, including 148 lines received from the International Crops Research Institute for the Semi-arid Tropics and 5 Korean cultivars (Dansusu 1ho, 2ho, 3ho, 4ho, and Muhanjaelae) received from the Rural Development Administration genebank were used in this study (
Supplementary Table S1). The seeds were planted in plots (3 × 15 m) and row spacing of 20 and 60 cm, respectively. Plants were grown in experimental fields at the Korea Atomic Energy Research Institute (KAERI, Jeongeup, Korea). Antioxidant compound contents were measured in eight selected plants.
Agronomic and morphological characteristics were investigated in 153 sweet sorghum plants planted at the end of May 2016. The following traits were measured: plant height (cm), panicle length (cm), stalk diameter (mm), leaf number (ea), leaf width (cm), leaf length (cm), sugar content (Brix), and tiller number (ea) post-harvest. Agronomic traits, such as ripening stage (days), seed yield (high; more than 1.0 ton/ha, middle; between 0.5 and 1.0 ton/ha), seed coat color, and seed- or panicle-specific characteristics, were further investigated in eight selected sorghum germplasms.
3-deoxyanthocyanin contents measurements
3-deoxyanthocyanin measurements were performed as described in
Awika et al. (2004) with some modifications. Analysis was done by ultra-high performance liquid chromatography (UPLC) (CMB-20A, Shimadzu Co., Kyoto, Japan) with gradient pump systems (LC-30AD, Shimadzu Co.) and a UV detector (SPD-M30A, Shimadzu). For UPLC analysis, grain powder samples (0.5 g) were extracted in 5 mL HCl:MeOH (1:99) at 20°C in the dark for 2 h, then centrifuged at 7000 ×
g for 10 minutes. The supernatant was filtrated through 0.45 μm membrane filters. Separation of the compounds was performed on an HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters Inc., MA, USA) using a linear gradient elution program with a mobile phase containing solvent A and solvent B. The flavonoid compounds were separated using the following gradients: 0–5 minutes, 10%–15% B; 5–10 minutes, 15%–20% B; 10–15 minutes, 20%–30% B; 15–20 minutes, 30%–50% B; 20–25 minutes, 50%–75% B; 25–30 minutes, 75%–100% B; 30–32 minutes, 100%-5% B; and 32–35 minutes, 5%-0% B; detection was at 480 nm and identification was made using commercial standards (
Awika et al. 2004;
Dykes et al. 2009).
The total tannin content was determined using the Folin–Ciocalteu method (Makkar
et al. 2000). The reaction mixture was prepared from a 1 mL aqueous solution of grain extract, with methyl alcohol (50%) and 1N Folin–Ciocalteu phenol reagent in distilled water. Samples were incubated at 25°C for 90 minutes, and the tannin absorbance level was measured using a UV-1800 spectrophotometer (Shimazuda Co.) at 725 nm. Standard curves were expressed as catechin equivalents. Total flavonoid measurements were measured as described by
Zhishen et al. (1999). A total of 1 mL aqueous solution of the extract was added to 4 mL distilled water in a 10 mL volumetric flask, and 0.3 mL 5% NaNO
2 was then added. After 5 minutes, 0.3 mL 10% AlCl
3 and 2 mL 1 N NaOH were added sequentially into the 10 mL flask, which was then filled up to 10 mL with distilled water. The absorbance level was compared with the prepared reagent blank at 510 nm.
Statistical analyses were performed with SPSS version 12 software (SPSS Institute, USA) using one-way analysis of variance followed by Duncan’s multiple range test. Differences were considered significant at P < 0.05.
RESULTS
Characterization of agronomic and morphological traits
Table 1 and
Supplementary Table S1 show the analysis of agronomic and morphological traits of 153 sweet sorghum germplasms. A variety of phenotypes were observed with respect to growth characteristics. Plant heights ranged from 70 (SS.22) to 430 (SS.99) cm, and panicle lengths ranged from 7.00 (SS.181) to 55.80 (SS.16) cm. Similarly, all leaf traits such as leaf number, leaf width, and leaf length exhibited significant variability (
Supplementary Table S1). Nine sweet sorghum plants showed more than 20 Brix, including SS.226 (21.6 Brix) and SS.7 (21.5 Brix). By contrast, the sugar content of 35 sweet sorghum germplasms could not be detected. SS.97 had the lowest recorded sugar content (4.5 Brix). With the exception of sugar content and tiller number, the measured agronomic traits were well-distributed among the 153 germplasms and closely followed a Gaussian distribution (
Fig. 1). These results indicated that the collected sweet sorghum germplasms used in this study did not show biased phenotypes from different genotypes.
Based on the agronomic trait results, we selected eight elite sweet sorghum germplasms that showed a high performance in terms of agronomic and growth characteristics. These included tall plant height (SS.74, dansusu 4ho), large panicle size with a short height (SS.117), wide stalk diameter (SS.174), high sugar content (SS.7, dansusu 2ho), seed-specific characteristics such as different seed color (SS.113), and a different type of ear head with seed coat color (SS.129). The eight selected plants also exhibited a high seed yield except for SS.7 (
Table 2).
3-deoxyanthocyanin compounds were analyzed in the eight selected sweet sorghum grains. The total 3-deoxyanthocyanin content, and levels of luteolinidin, apigeninidin, and apigeninidin glycosides were measured (
Fig. 2). SS.129 did not contain any 3-deoxyanthocyanins. A range of luteolinidin levels were detected among the remaining seven selected sweet sorghum grains, from 5.5 mg/100 g (dansusu 4ho) to 0.4 mg/100 g (SS.113) with an average of 3.7 mg/100 g. By contrast, apigeninindin contents were highest in SS.113 (24.9 mg/100 g) and lowest in dansusu 4ho (1.6 mg/100 g), with an average of 6.9 mg/100 g (
Table 3). Consistently, the pattern of total 3-deoxyanthocyanins contents ranged from 34.3 mg/100 g (SS.113) to 3.5 mg/100 g (dansusu 4ho). This result was also seen in high performance liquid chromatography (HPLC) 3D profiling (
Fig. 3).
The total flavonoid and tannin contents of the eight selected sweet sorghum grains are shown in
Fig. 4. Total tannin levels showed significant differences in levels, with SS.174 having the highest recorded content (5.0 mg/g), and SS.7 and dansusu 4ho having 2.4 mg/g and 2.8 mg/g, respectively (
Fig. 4A). The total flavonoid content was highest in SS.129 (7.52 mg/g) and lowest in dansusu 4ho (1.84 mg/g) with an average of 4.9 mg/g (
Fig. 4B). SS.129 had the highest level of total tannins and flavonoids, and dansusu 4ho had the lowest level of both compounds.
DISCUSSION
In this study, we investigated the agronomic traits of 153 sweet sorghum germplasms. Based on this analysis, we selected eight elite sweet sorghum grains to determine their biochemical composition. The agronomic traits revealed a variety of phenotypes with respect to plant height, panicle length, and stalk diameter, as well as leaf traits such as number, width, and length (
Fig. 1,
Supplementary Table S1).
The stalk of sweet sorghum contains large amounts of fermentable sugar, and is a profitable alcoholic material for the production of bioethanol (
Laopaiboon et al. 2009;
Mathur et al. 2017). High yields of biomass can be converted into more accessible energy forms (
Lipinsky 1978), so the observed higher sugar content in sweet sorghum is beneficial to the rate of bioethanol production. The sugar content of sweet sorghum is measured in Brix units, which represent the percent of soluble sugars. For example, one degree Brix is equal to 1 g of sugar per 100 g of juice. It was previously reported to vary widely. For example,
Kumar et al. (2013) found it to range from 6.0–15.0 Brix among 19 sweet sorghum genotypes, while some sweet sorghum lines were reported to have a maximum value of 23.0 Brix (
Srinivasa Rao et al. 2009). Our identification of nine sweet sorghum stalks with more than 20 Brix (
Supplementary Table S1) will be a useful resource of bioethanol for the biofuel industry and fermentable sugar products.
We investigated the composition of 3-deoxyanthocyanins, total flavonoids, and the tannin content for potential antioxidant properties in selected sweet sorghum. Luteolinidin and apigeninidin belong to the 3-deoxyanthocyanins and accumulate in the stalk, grain, leaf, and glume of sorghum plants (
Awika and Rooney 2004;
Kayodé et al. 2011). These compounds are not commonly found in higher plants (
Clifford 2000). Apigeninidin has well-documented human health benefits associated with its antioxidant activity (
Yang et al. 2009;
Woo et al. 2012). Previous studies reported that the 3-deoxyanthocyanin content was determined by the grain color and secondary plant color in sorghum plants (
Awika et al. 2004;
Dykes et al. 2005). Our HPLC 3D profiling and HPLC analysis (
Table 3,
Fig. 3) revealed that total 3-deoxyanthocyanin and apigeninidin contents of SS.113, especially in the whole grain, were 3–9 fold higher than in other plants with a gold-yellow grain color (
Table 3). This supports the finding that the 3-deoxyanthocyanin content is affected by grain color (
Table 2). The total flavonoid content was separated into two groups: group 1 had an elevated content of more than 5 mg/g (dry weight), and included SS.113, SS.129, SS.117, SS.74, and dansusu 4ho, while group 2 had a low flavonoid level of below 3 mg/g (dry weight), and included dansusu 2ho, SS.174, and SS.7. Interestingly, SS.129 had the highest total flavonoid level (7.52 mg/g), but no detectable 3-deoxyanthocyanins compounds.
Tannins are the most important biochemical compounds of sorghum, with well-known antioxidant properties (
Chung et al. 1998;
Awika and Rooney 2004). The tannin content of selected sweet sorghum in this study ranged from 5.0 to 2.4 mg/g, which was lower than values reported by
Jambunathan and Mertz (1973) of more than 10 mg/g in hightannin sorghum plants.
In conclusion, we performed agronomic traits analysis in 153 sweet sorghum plants, and selected eight elite lines based on these data. The selected sweet sorghum plants had useful agronomic and growth characteristics such as taller plant height, large panicle size with short height, high sugar content, and seed-specific characteristics in a temperate climate. Our agronomic trait and antioxidant data are valuable for basic research in sweet sorghum breeding.
Supplementary Information
ACKNOWLEDGEMENTS
This work was supported by the research program of KAERI, Republic of Korea. We thank Sarah Williams, PhD, from Edanz Group (
www.edanzediting.com) for editing a draft of this manuscript.
Fig. 1Gaussian distribution of agronomic traits among 153 sweet sorghum plants. Data are measured values of each genotype.
Fig. 2High performance liquid chromatography (HPLC) profiles of sweet sorghum 3-deoxyanthocyanins detected at 480 nm. Peaks 1 and 2 are luteolinidin and apigeninidin, respectively. Peak 3 was tentatively identified as glycosides of apigeninidin. Peaks were identified on the basis of matching retention times and spectral characteristics with commercial standards. (A) Dansusu 4ho. (B) SS174. (C) SS113. (D) SS7. (E) SS117. (F) SS74. (G) Dansusu 2ho.
Fig. 3High performance liquid chromatography (HPLC) 3D profiles of sweet sorghum 3-deoxyanthocyanins detected at 480 nm. (A) Dansusu 4ho. (B) SS174. (C) SS113. (D) SS7. (E) SS117. (F) SS74. (G) Dansusu 2ho.
Fig. 4Tannin content and total flavonoid content in the germplasm of selected sweet sorghum. *Superscript letters indicate significant differences at the 5% level (Duncan’s multiple range tests). (A) Tannin content (mg/g). (B) Total flavonoid content (mg/g).
Table 1Summary of agronomic characteristics among 153 sweet sorghum germplasms.
Table 1
|
Values |
Plant height (cm) |
Panicle length (cm) |
Stalks diameter (mm) |
Leaf number (ea) |
Leaf width (cm) |
Leaf length (cm) |
Sugar content (brix) |
Tiller number (ea) |
|
Min |
70.00 |
7.00 |
11.60 |
5.00 |
4.00 |
38.00 |
4.50 |
1.00 |
|
Mean |
262.48 |
26.66 |
24.26 |
11.76 |
8.45 |
71.53 |
15.29 |
2.48 |
|
Max |
430.00 |
55.80 |
38.40 |
25.00 |
14.00 |
121.00 |
21.60 |
10.00 |
|
Line No. |
|
Min |
SS.22 |
SS.181 |
SS.180 |
SS.22 |
SS.10 |
SS.224 |
SS.97 |
SS.2 |
|
Max |
SS.99 |
SS.16 |
SS.174 |
SS.21 |
SS.68 |
SS.134 |
SS.226 |
SS.124 |
Table 2Agronomic and morphological traits in eight selected sweet sorghum plants.
Table 2
|
No. |
Lines |
Plant height (cm) |
Panicle length (cm) |
Stalks diameter (mm) |
Leaf number (ea) |
Leaf width (cm) |
Leaf length (cm) |
Sugar content (brix) |
Tiller number (ea) |
Ripen stage (days) |
Seed yield |
Seed coat color |
Panicle/seed Characteristics |
|
1 |
Dansusu 4ho |
397.7 |
38.7 |
28.7 |
9.3 |
9.5 |
81.3 |
15.5 |
3 |
65 |
high |
Black |
|
|
2 |
SS.174 |
307 |
27.7 |
38.4 |
11.3 |
10 |
80.3 |
17.2 |
1 |
80 |
high |
Black |
|
|
3 |
SS.113 |
375 |
30 |
30.7 |
12 |
10 |
96 |
17.2 |
1 |
100↑ |
high |
Black |
Gold-yellow seed color |
|
4 |
SS.129 |
379 |
31 |
34.6 |
12 |
10.5 |
90 |
11.3 |
1 |
100↑ |
high |
White |
Goose neck |
|
5 |
SS.7 |
315 |
30 |
34.5 |
19 |
11 |
90 |
21.5 |
1 |
100↑ |
middle |
Black |
Compact type |
|
6 |
SS.117 |
280 |
38.7 |
22.3 |
8.7 |
9.5 |
102.3 |
13.5 |
1 |
65 |
high |
Dark-black |
Long-round head |
|
7 |
SS.74 |
407 |
21 |
23.2 |
10 |
9 |
67 |
ND |
1 |
100↑ |
high |
Brawn |
|
|
8 |
Dansusu 2ho |
256.7 |
30 |
26.8 |
12.3 |
9.3 |
77.7 |
20 |
2 |
72 |
high |
Red-black |
|
Table 33-deoxyanthocyanin levels in the whole grain of eight selected sweet sorghum plants.
Table 3
|
Cultivar name |
Luteolinidin (mg/100 g) |
Apigeninidin (mg/100 g) |
Glycosides of Apigeninidinz) (mg/100 g) |
Total 3-deoxyanthocyanins |
|
Dansusu 4ho |
1.4 ± 0.2c
|
1.6 ± 0.2b
|
0.5 ± 0.1c
|
3.5 |
|
SS.174 |
3.7 ± 0.8b
|
5.1 ± 0.8b
|
0.8 ± 0.1c
|
9.6 |
|
SS.113 |
0.4 ± 0.0c
|
24.9 ± 7.1a
|
9.1 ± 1.7a
|
34.3 |
|
SS.129 |
- |
- |
- |
- |
|
SS.7 |
1.4 ± 0.2c
|
1.7 ± 0.2b
|
0.5 ± 0.1c
|
3.6 |
|
SS.117 |
5.6 ± 1.2b
|
4.7 ± 0.1b
|
1.1 ± 0.0b
|
11.4 |
|
SS.74 |
8.2 ± 1.9a
|
5.7 ± 0.7b
|
1.4 ± 0.1b
|
15.3 |
|
Dansusu 2ho |
5.5 ± 1.1b
|
4.4 ± 0.2b
|
1.1 ± 0.0b
|
11.0 |
|
Mean |
3.7 |
6.9 |
2.1 |
12.7 |
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