Breeding Black Soybeans for High Yield and First Pod Height Is a Promising Approach to Improving Thai Commercial Soybean Varieties

Abstract

Black soybeans are rich in beneficial substances like anthocyanins, which help combat free radicals, and also have a high protein content. However, the soybean production system in Thailand struggles with issues relating to the use of machinery for harvesting. For efficient harvesting with machinery, the first pod of the soybean cultivar should be positioned at a height greater than 10 cm. Thailand has not yet reported black soybean cultivars with the height of the first pod measuring 10–15 cm above the ground. Therefore, the aim of this experiment was to improve the commercial soybean varieties KKU35, SJ5, NSW1, and CM60 in Thailand by increasing their first pod height and developing black grains through crossbreeding with the KKUSB–108 soybean germplasm. Subsequently, the pedigree selection method was used to evaluate and select plants with black grains, good growth performance, and a first pod height exceeding 10 cm from the F₂ to F₅ generations. The selected line of F₅ was selected, while the grains of the F₆ generation were designated as recombinant inbred lines (RILs). Eight soybean RILs, namely KKU35xKKUSB–108–12–4–3, KKU35xKKUSB–108–24–5–7, SJ5xKKUSB–108–25–2–1, SJ5xKKUSB–108–30–3–7, NSW1xKKUSB–108–49–3–3, NSW1xKKUSB–108–49–3–6, CM60xKKUSB–108–41–1–7, and CM60xKKUSB–108–64–4–8, together with the Sukhothai 3 black soybean commercial variety, were laid out in a randomized complete block design (RCBD) with three replications at the Agronomy Field Crop Station, Khon Kaen University, over two seasons: the rainy season in 2021 and the dry season in 2022. The results revealed that the first pod height of all RILs in the rainy and dry seasons was higher than Sukhothai 3. The RILs showing a consistently high yield in both the rainy and dry seasons were SJ5xKKUSB-108-25-2-1 (1.85 and 1.86 T/ha), SJ5xKKUSB-108-30-3-7 (1.65 and 1.72 T/ha), NSW1xKKUSB-108-49-3-6 (1.52 and 1.83 T/ha), and CM60xKKUSB-108-64-4-8 (1.60 and 1.61 T/ha). Moreover, the RIL, NSW1xKKUSB-108-49-3-6, has a protein content of up to 44.21% in the dry season and shorter maturity than other RILs. This RIL can be used for cropping rotation systems in areas with limited time and water resources. This work provides a resource of black soybean RILs with high yield and first pod height for soybean breeding programs in the future. However, yield and protein content were affected by season (S), genotype (G), and the S × G interaction, indicating that RILs require a diverse environment for regional yield trials in the future.

1. Introduction

Soybean (Glycine max (L.) Merrill) is an economically significant leguminous crop. It has been cultivated in China for over 6000 years [1]. Several soybean varieties have subsequently been developed and adopted to grow in different geographic regions worldwide [2]. The soybean grain contains high amounts of protein (39.97–41.45%), carbohydrates (30.35–32.80%), lipids (13.41–19.42%), dietary fiber, vitamins (B, C, and E), and minerals (Na, K, P, and Fe) [3]. Currently, soybeans are a global commodity, with 170 countries trading this crop internationally, representing an estimated total value of USD 155 billion in 2024 [4]. Soybeans predominantly provide oil and protein sources for food and animal feed worldwide [5]. An abundance of accumulated pigment in the seed coats results in different-colored soybeans, such as black, brown, green, and yellow soybeans (YS); among them, YS is the most popular [6].

Black soybeans are also an excellent food source for disease prevention and contain health-promoting substances. Takahashi et al. [7], Khosravi and Razavi [8], and Nirmal et al. [9] found that black soybeans had the highest antioxidant activity compared to others with colored seed coats. Anthocyanins, a group of reddish or purple flavonoids, are reported to be the primary pigments in the black soybean varieties [10,11]. They are abundant in the seed coats of black soybean varieties and have health-promoting benefits, including antioxidant effects, reduced cholesterol, anticancer properties, enhanced immunity, and mitigating the effects of aging [12]. Soybeans have been widely consumed as nutritional sources of protein and raw material for oriental medicine for centuries in Asia [13], in countries such as China, Japan, Korea, Indonesia, and India [14]. Black soybeans have been utilized in traditional Chinese medicine for blood improvement, detoxification, and anti-inflammatory properties [15]. In Korea, medium and large black soybean seeds are used in food processing, such as side dishes or mixed with steamed rice [16]. However, Indonesia uses them as a traditional holy food in various cuisine preparations and domestic and large-scale industries [17]. Black soybeans are primarily used as the raw material for soy sauce because they give it a natural black color and delicious taste [18,19]. Many traditional soy foods currently consumed in Asian countries, including tofu, tempeh, and natto, are known to be healthy foods [20]. Anthocyanins accumulated in the seed coat’s epidermal palisade layer could be isolated into anthocyanin-rich fractions for application as functional colorants or food components in the food and feed industries [21,22,23].

Since 1999, “Sukhothai 3” has been the only commercial variety of black soybean to be released in Thailand. Until now, the planting areas of black soybeans in Thailand have not been reported. They have traditionally been cultivated on a small scale in isolated areas throughout the northern and central regions. As previously mentioned, the advantages of black soybeans have resulted in high prices. The Royal Project Shops in Thailand sell black soybean grain for USD 2 per 400 g [Chankaew, personal observation]. Farmers need to promote and extend black soybean production to increase their incomes. However, the limitations of the black soybean variety, the high production costs caused by the aging society of soybean growers, and the lack of advanced mechanized technology in soybean production might limit production progress [24]. The harvesting process is considered one of the most labor-intensive operations. Therefore, the use of combined harvesters may provide a solution to this problem [25]. Investing in modern machinery can enable tasks to be performed more efficiently and reduce the need for manual labor, especially for harvesters, saving time while significantly improving the profitability of soybean production. However, soybean varieties in Thailand have a low first pod height, resulting in a significant yield loss when a combined harvester is used.

The breeding of black grain soybeans from the past to the present has involved developing varieties with high nutritional value that respond to the needs of the industrial, medical, or pharmaceutical industries [26]. Classical genetics demonstrate that multi-loci, at least five genetic loci (I, R, T, W1, and O), are involved in the flavonoid-based pigmentation process of soybeans. Among them, the I, R, and T loci are involved in the biosynthesis of the pigments; thus, the combination of these genes results in specific colors, such as black (i, R, T), imperfect black (i, R, t), brown (i, r, T), or buff (i, r, t) [27]. On the other hand, only the recessive alleles background of i, r or i, t are influenced by O and W1 in the pigmentation [28]. This information demonstrates that the genetic background and gene combination are involved in the seed coat color of soybeans. On the other hand, black grain soybeans can pass their genetics on to offspring through a single recessive gene [29]. Moreover, the traits that meet agricultural practice and production methods should be considered, such as plant height, first pod height, and yield [30,31]. The first pod height (FPH) is a quantitative characteristic [32,33]. It is a trait of crucial importance when using a mechanical combine harvester [34]. Mechanized soybean harvesting is a reliable and cost-effective option for farmers. To minimize seed yield loss from pods below the cutter level, the FPH of soybeans must be reasonably high, at least 12 cm [35]. Consequently, breeding soybeans with black seeds and high FPH is an essential breeding goal. Genetics plays a significant role in the soybean pod height set. The mean heritability value of FPH was 66% across three F₂ populations; therefore, further genetic selection for pod height must be conducted carefully to avoid seed yield loss [36]. The high heritability estimates may help in early-stage phenotypic selection for FPH [37]. Jiang et al. [32] identified several candidate genes associated with plant growth. These genes, which are involved in the pathways of auxin response factor 9, serine/threonine-protein kinase, and transmembrane amino acid transporter family protein, exhibited high expression levels in soybean stems. Although molecular-based plant breeding methods and techniques, such as marker-assisted selection, are part of their cultivar breeding program, soybean breeders also successfully develop cultivars utilizing conventional breeding approaches, such as single seed descent and pedigree methods [38]. The pedigree approach is required to make selections over vast F₂ generations for screening to ensure the desired recombinant inbred lines (RILs) are obtained. In succeeding segregating generations, their offspring are evaluated in the intergenerational population (F₅–F₆). This method allows breeders to improve soybean varieties gradually over successive generations. Focusing on specific traits can enhance yield, adaptability, and quality [39].

Therefore, this study aimed to develop a Thai black soybean with an FPH of 10–15 cm from the ground to make it suitable for mechanical harvesters. To achieve this aim, the pedigree selection method was used to select the RILs from crossbreeding among the commercial yellow soybean varieties and the black soybean germplasm. The goal was also to increase the value of black soybeans for farmers and generate a black soybean germline source with a high FPH for Thailand’s future black soybean breeding program.

2. Materials and Methods

2.1. Plant Materials

Four Thai commercial soybean varieties, KKU35, SJ5, NSW1, and CM60, and one black soybean germplasm, KKUSB-108, were used as parental lines. All Thai soybean varieties had yellow seeds with high yield and stability, commercially cultivated through soybean plantations in Thailand. The black soybean germplasm KKUSB-108 had a high FPH. The commercial black soybean variety Sukhothai 3 was also used as a check variety in preliminary yield trials.

2.2. Population Development

Five parental varieties, KKU35, SJ5, NSW1, CM60, and KKUSB-108 were used for four bi-parental crosses (KKU35xKKUSB-108, SJ5xKKUSB-108, NSW1xKKUSB-108, and CM60xKKUSB-108), and generated the recombinant inbred lines (RILs), F₆ population. Briefly, 5–10 F₁ seeds from each cross were generated and then a single F₁ from each was self-fertilized to generate F₂ seeds. The 180 plants of the F₂ populations of each cross were planted in the experimental block. Ten F₂ plants of each cross with black seeds, good growth performance, and an FPH of more than 10 cm were selected for the F₃ generation. The 10 F₃ populations of each cross were planted in an experimental field with Sukhothai 3 and their parents. Ten F₃ plants of each cross with good agronomical performance and an FPH of more than 10 cm were selected for the F₄ generation. The 10 F₄ populations of each cross were planted in an experimental field with Sukhothai 3 and their parents. Five F₄ plants of each cross with good performance and an FPH of more than 10 cm were selected for the F₅ generation. The 5 F₅ populations of each cross were planted in an experimental field with Sukhothai 3 and their parents. Two F₅ plants of each cross with good performance and an FPH of more than 10 cm were selected for the F₆ generation. After the F₆ generation, the RIL_S with just the name and cross details were used for the preliminary yield trial at the research station with Sukhothai 3 used as a check variety (Figure 1).

2.3. An F₂ to F₅ Experimental Block and Fields

The F₂ populations of each cross were planted in the experimental block during 2020. The experimental block size was 5 m × 1 m with a spacing of 5 cm between plants and 50 cm between rows. Fertilizer was initially applied at 14.06 kg/ha (N₂-P₂O₅-K₂O) 15 days after planting (DAP). In addition, a second fertilizer application was applied, consisting of 18.75 kgN₂/ha, 37.50 kgP₂O₅/ha, and 18.75 kgK₂O/ha at 60 DAP. Herbicides and insecticides were used as necessary, as recommended by Thailand’s DOA, and irrigation was practiced throughout the experimental period as required. Each cross of the F₃ to F₅ populations was planted in the experimental field from 2020 to 2021. The experimental units consisted of three parallel 4 m long rows per line, spacing of 25 cm between holes and 50 cm between rows, and 17 holes per row. According to local practices, sowing took place from July to August in the rainy season and December to January in the dry season. At 15 DAP, plants were thinned to three plants per hole, with the weeds controlled by hand weeding. Fertilizer was initially applied at 14.06 kg/ha (N₂-P₂O₅-K₂O) at 15 DAP, and the second fertilizer application at 18.75 kgN₂/ha, 37.50 kgP₂O₅/ha, and 18.75 kgK₂O/ha at 60 DAP. Herbicides and insecticides were used as necessary, as recommended by Thailand’s DOA, and irrigation was practiced throughout the experimental period as required.

2.4. Preliminary Yield Trials

The first yield trial was conducted during the rainy season of 2022 from July to October (planting date 27 July 2022) at the Agronomy Field Crop Station, Khon Kaen University, Thailand. The second yield trial was conducted during the rainy season of 2022–2023, with the experimental period ranging from December 2022 to April 2023 (planting on 7 December 2022) at the Agronomy Field Crop Station, Khon Kaen University, Thailand. Eight RILs and the Sukhothai 3 check varieties were subjected to the randomized complete block design (RCBD), with three replicates of plot sizes 4 × 1.5 m and plant spacing of 25 cm between holes and 50 cm between rows. At 15 DAP, plants were thinned to three per hole, and weeds were controlled by hand weeding. Fertilizer was initially applied at 14.06 kg/ha (N₂-P₂O₅-K₂O) at 15 DAP, with a second fertilizer application at 18.75 kgN₂/ha, 37.50 kgP₂O₅/ha, and 18.75 kgK₂O/ha at 60 DAP. Herbicides and insecticides were used to eliminate diseases and insects as outbreaks occurred, as recommended by Thailand’s DOA. At the same time, irrigation was practiced throughout the experimental period with a sprinkler system as required.

2.5. Data Collection

The growth, yield, and yield components of black soybean varieties were recorded according to Srithongtae et al. [24]. The height of a soybean plant was measured from the ground level to the highest part at 50% flowering (50% of the plants in each plot showed at least one fully bloomed flower) and full maturity stages. The height of the first pod was recorded as the full maturity stage. The data were recorded by randomly measuring ten soybean plants per subplot. The number of days for 50% flowering and full maturity stages were also recorded. After harvesting, data on the number of nods per plant (nods/plant) and grains per plant (grains/plant) were randomly collected for ten plants/subplots. The weight of 100 grains was also recorded by weighing the 100 represented grains in each subplot. The grain yield was weighed in each subplot.

A percentage of the protein content was determined. The amount of nitrogen (N) components in soybean grain samples was measured using a nitrogen analyzer (Leco FP828 Nitrogen Analyzer, LECO Corporation, Saint Joseph, MI, USA) at the Animal Nutrition Unit, Department of Animal Science, Khon Kaen University. The purpose of this was to evaluate the crude protein content. In summary, the soybean grain was dried for seven days at 50 °C. Following a thorough grinding of the material, 0.3 g of pure tin foil was weighed, put into a tin capsule, and then placed into a sample loader. Using (N × 6.25) as a formula, the crude protein content (%) was determined [40].

2.6. Data Analysis

A combined analysis of variance (ANOVA) for a randomized complete block design (RCBD) through each environment was carried out for growth, yield, yield components, and protein content using the Statistix 10^© program version 10.0 (1985–2013) (Analytical Software, Tallahassee, FL, USA). The mean of each trait was compared using Tukey’s Honestly Significant Difference (HSD) via R-Stat [41].

3. Results

3.1. Population Development

The 8 RILs of black soybean with high first pod height consisting of KKU35xKKUSB–108–12–4–3, KKU35xKKUSB–108–24–5–7, SJ5xKKUSB–108–25–2–1, SJ5xKKUSB–108–30–3–7, NSW1xKKUSB–108–49–3–3, NSW1xKKUSB–108–49–3–6, CM60xKKUSB–108–41–1–7, and CM60xKKUSB–108–64–4–8 were successfully bred through crossbreeding from the commercial yellow soybean varieties KKU35, SJ5, NSW1, and CM60 with the KKUSB–108 black soybean germplasm using the pedigree selection method (

Table 1. Details of the soybean varieties and recombinant inbred lines used in this study.

No.	Genotypes	Type of Varieties	Maturity Type	Sources	Grain Color
1	KKU35	Check variety	Late	Khon Kaen University	Yellow
2	SJ5	Check variety	Intermediate	Department of Agriculture, Thailand	Yellow
3	NSW1	Check variety	Early	Department of Agriculture, Thailand	Yellow
4	CM60	Check variety	Intermediate	Department of Agriculture, Thailand	Yellow
5	Sukhothai 3	Check variety	Intermediate	Department of Agriculture, Thailand	Black
6	KKUSB–108	Check variety	Intermediate	Khon Kaen University	Black
7	KKU35xKKUSB–108–12–4–3	Breeding line	Late	Khon Kaen University	Black
8	KKU35xKKUSB–108–24–5–7	Breeding line	Late	Khon Kaen University	Black
9	SJ5xKKUSB–108–25–2–1	Breeding line	Intermediate	Khon Kaen University	Black
10	SJ5xKKUSB–108–30–3–7	Breeding line	Intermediate	Khon Kaen University	Black
11	NSW1xKKUSB–108–49–3–3	Breeding line	Early	Khon Kaen University	Black
12	NSW1xKKUSB–108–49–3–6	Breeding line	Early	Khon Kaen University	Black
13	CM60xKKUSB–108–41–1–7	Breeding line	Intermediate	Khon Kaen University	Black
14	CM60xKKUSB–108–64–4–8	Breeding line	Intermediate	Khon Kaen University	Black

).

3.2. Combined ANOVA

The results revealed that the principal effect of the season (S) was significant for days to 50% flowering, days to full maturity, node/plant, grain/plant, weight of 100 grains, yield, and protein content, while the genotype (G) was significant for all traits. The S × G interaction was significant for plant height at 50% flowering, days to full maturity, node/plant, grain/plant, yield, and protein content (

Table 2. Mean squares from the combined analyses of growth data, yield, and yield components of soybean genotypes grown in the rainy and dry seasons of 2022–2023 at Khon Kean University, Thailand.

Source of Variance	df	Growth Data onSoybean Genotypes
		Plant Height at 50% Flowering	Days to 50% Flowering	Plant Height at Full Maturity	Days to Full Maturity	First Pod Height
Season (S)	1	158.03 ns	824.46 **	91.68 ns	12.51 *	0.36 ns
Error S × rep (R)	4	17.56	5.90	68.96	0.64	2.31
Genotype (G)	8	429.80 **	31.71 *	1010.95 **	170.12 **	37.80 **
S × G	8	181.72 **	15.38 ns	79.15 ns	36.14 **	2.39 ns
Error S × R × G	32	30.42	10.61	74.54	1.75	1.45
Total	53
Source of Variance	df	Yield and Yield Componentsof Soybean Genotypes
		Node/Plant	Seed/Plant	Weight of 100Grains	Yield	Protein Content
Season (S)	1	72.98 **	1157.87 *	14.36 *	0.6556 **	362.58 **
Error S × rep (R)	4	2.27	112.82	1.71	0.0057	0.075
Genotype (G)	8	86.79 **	726.41 **	14.06 **	0.3856 **	24.24 **
S × G	8	64.49 **	999.40 **	2.70 ns	0.0463 **	5.56 **
Error S × R × G	32	6.07	74.49	1.73	0.0054	0.46
Total	53

** = significantly different at p < 0.01, * = significantly different at p < 0.05, ^ns = non-significant at p > 0.05. df = degree of freedom.

). The results indicated that plant height at full maturity and the FPH of each variety were stable during the different seasons. Consequently, these traits were selected and fixed during population development. Yield and protein content were affected by all components (S, G, and S × G), implying the variability of these traits based on S, G, and S × G factors (Table 2), indicating that selective breeding requires a diverse environment for the regional yield trial step.

3.3. Growth Data on Black Soybean Genotypes

The combined mean values for plant height at 50% flowering, days to 50% flowering, plant height at full maturity, days to full maturity, FPH, node/plant, seed/plant, weight of 100 grains, yield, and protein content across two field seasons of the black soybean RILs and Sukhothai 3 check variety are presented in

Table 3. Growth data on soybean genotypes, including plant height at 50% flowering, days to 50% flowering, plant height at full maturity, days to full maturity, and FPH of soybean genotypes grown during the rainy and dry seasons of 2022–2023 at Khon Kean University, Thailand.

Varieties/Lines	Growth Data on Soybean Genotypes
	Plant Height at 50% Flowering (cm)		Days to 50% Flowering (Day)		Plant Height at Full Maturity (cm)		Days to Full Maturity (Day)		First Pod Height (cm)
	Rainy	Dry	Rainy	Dry	Rainy	Dry	Rainy	Dry	Rainy	Dry
KKU35xKKUSB–108–12–4–3	63.97 b	68.54 ab	45.00 a	49.00	81.54 ab	81.21 a	98.67 ab	107.00 a	10.96 bc	12.11 a
KKU35xKKUSB–108–24–5–7	80.50 a	66.39 ab	41.00 abc	48.66	88.47 a	88.52 a	100.00 a	104.33 a	15.02 a	12.81 a
SJ5xKKUSB–108–25–2–1	85.89 a	72.11 a	41.00 abc	47.66	94.18 a	84.73 a	99.00 ab	91.67 bc	11.65 b	12.38 a
SJ5xKKUSB–108–30–3–7	60.68 b	69.29 ab	43.00 ab	48.66	73.48 ab	67.27 abc	98.67 ab	91.00 bc	10.59 bc	9.67 a
NSW1xKKUSB–108–49–3–3	53.53 b	66.42 ab	37.00 c	45.33	60.96 b	74.23 ab	89.00 e	87.00 c	10.55 bc	12.74 a
NSW1xKKUSB–108–49–3–6	54.53 b	55.34 ab	37.00 c	44.33	54.89 b	56.54 bc	87.33 e	86.67 c	10.85 bc	10.43 a
CM60xKKUSB–108–41–1–7	54.78 b	67.95 ab	39.00 abc	51.00	81.96 ab	75.36 ab	94.33 d	94.33 b	9.31 c	9.90 a
CM60xKKUSB–108–64–4–8	54.49 b	69.99 ab	39.00 bc	53.00	79.88 ab	73.94 ab	97.00 bc	95.67 b	9.27 c	9.59 a
Sukhothai 3	50.41 b	53.53 b	39.00 abc	44.33	58.27 b	48.37 c	95.00 cd	93.00 b	4.82 d	4.87 b
Mean	62.09	65.51	40.18	48.00	74.85	72.24	95.44	94.51	10.33	10.50
F-test	*	**	*	ns	*	*	*	*	*	*
CV%	7.84	9.30	5.08	8.61	12.62	10.70	0.93	1.96	5.90	15.18

** = significantly different at p < 0.01, * = significantly different at p < 0.05, ns = non-significant at p > 0.05. Different letters after the mean within a column indicate a significant difference using Tukey’s honestly significant difference (HSD). CV = coefficient of variation.

. The results show that the RILs and Sukhothai 3 varieties significantly differ in all evaluated traits except days to 50% flowering during the dry season (Table 3).

The mean values for plant height at 50% flowering of RILs during the rainy and dry seasons ranged from 53.53–85.89 cm and 55.34–72.11 cm. The RILs’ KKU35xKKUSB–108–24–5–7 and SJ5xKKUSB–108–25–2–1 were taller than those of Sukhothai 3, which were 50.41 and 53.53 cm, respectively (Table 3). The mean values for days to 50% flowering during the rainy season for RILs ranged from 37.00 to 45.00 days, comparable with Sukhothai 3, which was 39.33 days, while during the dry season, the days to 50% flowering showed no significant difference (Table 3). The mean values for plant height at full maturity of RILs during the rainy and dry seasons ranged from 54.89 to 94.18 cm and 56.54 to 88.52 cm. The values for RILs, KKU35xKKUSB–108–24–5–7, and SJ5xKKUSB–108–25–2–1 were taller than those of Sukhothai 3 in the rainy season, and the values for KKU35xKKUSB–108–12–4–3, KKU35xKKUSB–108–24–5–7, SJ5xKKUSB–108–25–2–1, NSW1xKKUSB–108–49–3–3 CM60xKKUSB–108–41–1–7, and CM60xKKUSB–108–64–4–8 were taller than those of Sukhothai 3 in the dry season, which were 58.27 and 48.37 cm, respectively. The results differed slightly from the plant height data for 50% of flowering. The RILs from the KKU35 parent showed a taller plant at full maturity than the RILs from the NSW1 parent and Sukhothai 3 check variety during the rainy season, whereas, during the dry season, almost all RILs were taller than Sukhothai 3 (Table 3). The results indicate that the critical success in selecting RILs with a high-value plant height at 50% flowering and full maturity of black soybean RILs may depend on the height of Thai commercial parental varieties.

The RILs from the NSW1 parent, NSW1xKKUSB–108–49–3–3, and NSW1xKKUSB–108–49–3–6 exhibited shorter maturity than all RILs from other parents and Sukhothai 3, with the mean values for days to full maturing ranging from 87.33 to 89.00 days and 86.67 to 87.00 days during the rainy and dry seasons, respectively (Table 3). The mean values for FPH of RILs during the rainy and dry seasons ranged from 9.27 to 15.02 cm and 9.59 to 12.81 cm, taller than those of Sukhothai 3 (4.82 and 4.87 cm), respectively (Table 3). The results indicate that the FPH of black soybean RILs was twice as much as that of Sukhothai 3, making them suitable for a mechanical combine harvester.

3.4. Yield, Yield Components, and Protein Content of Black Soybean Genotypes

The mean values for node/plant of RILs during the rainy and dry seasons ranged from 13.91 to 31.39 nodes and 13.71 to 28.23 nodes, comparable to the values for Sukhothai 3, which were 16.58 and 15.20 nodes, respectively. Of the RILs, SJ5xKKUSB–108–25–2–1 had more nodes/plants than Sukhothai 3 during rainy season, while all RILs except KKU35xKKUSB–108–12–4–3 had more nodes/plants than Sukhothai 3 during dry season (

Table 4. Yield and yield components, including node/plant, grain/plant, weight of 100 grains, yield, and protein content of soybean genotypes grown during the rainy and dry seasons of 2022–2023 at Khon Kean University, Thailand.

Varieties/Lines	Yield and Yield Components of Soybean Genotypes
	Node/Plant		Grain/Plant		Weight of 100 Grains (g)		Yield (T/ha)		Protein Content (%)
	Rainy	Dry	Rainy	Dry	Rainy	Dry	Rainy	Dry	Rainy	Dry
KKU35xKKUSB–108–12–4–3	18.76 b	13.71 f	123.44 b	106.43 de	11.28 ab	11.86 ab	1.40 de	1.68 ab	38.71 a	42.30 abc
KKU35xKKUSB–108–24–5–7	20.94 b	22.63 cd	120.72 b	131.6 bc	10.12 b	9.86 b	1.46 cd	1.77 ab	34.245 b	40.31 bcd
SJ5xKKUSB–108–25–2–1	31.39 a	22.89 c	156.11 a	116.9 cde	9.85 b	9.83 b	1.85 a	1.86 a	30.16 c	41.05 abcd
SJ5xKKUSB–108–30–3–7	17.26 b	25.00 b	105.03 b	118.9 cd	11.85 ab	14.84 a	1.65 b	1.72 ab	37.525 a	43.75 a
NSW1xKKUSB–108–49–3–3	16.08 b	28.23 a	101.04 b	154.33 a	11.87 ab	13.93 ab	1.30 e	1.72 ab	35.21 b	42.75 ab
NSW1xKKUSB–108–49–3–6	13.91 b	21.51 d	105.35 b	127.9 bc	13.18 a	14.33 ab	1.52 bcd	1.83 ab	38.64 a	44.25 a
CM60xKKUSB–108–41–1–7	23.30 ab	25.49 b	115.77 b	136.53 b	12.54 a	15.23 a	1.46 cd	1.57 b	35.065 b	38.70 d
CM60xKKUSB–108–64–4–8	20.98 b	25.45 b	122.35 b	134.57 b	12.66 a	13.68 ab	1.60 bc	1.61 ab	34.355 b	39.35 cd
Sukhothai 3	16.58 b	15.20 e	97.10 b	103.09 e	12.66 a	11.74 ab	1.44 f	1.66 c	30.015 c	38.60 d
Mean	19.91	22.23	116.32	125.58	11.78	12.81	1.44	1.66	34.88	41.22
F-test	*	*	*	*	*	*	*	*	*	*
CV%	17.37	1.92	9.55	4.04	6.34	13.32	3.6	5.44	1.53	1.95

* = significantly different at p < 0.05. Different letters after the mean within a column indicate a significant difference using Tukey’s honestly significant difference (HSD). CV = coefficient of variation.

). The mean values for the grains/plants of RILs during the rainy and dry seasons ranged from 101.04 to 156.11 grains and 106.43 to 154.33 grains, comparable to those of Sukhothai 3, which were 97.10 and 103.09 grains, respectively. Of the RILs, SJ5xKKUSB–108–25–2–1 had more grains/plants than Sukhothai 3 in the rainy season. On the other hand, all RILs except KKU35xKKUSB–108–12–4–3 and SJ5xKKUSB–108–25–2–1 had more grains/plants than Sukhothai 3 during the dry season (Table 4). The mean values for the weight of 100 grains of RILs during the rainy and dry seasons ranged from 9.85 to 13.18 g and 9.83 to 14.84 g, comparable to those of Sukhothai 3, which were 12.66 and 11.74 g, respectively (Table 4). The mean values for the yield of RILs during the rainy and dry seasons ranged from 1.30 to 1.85 T/ha and 1.57 to 1.86 T/ha higher than those of Sukhothai 3, which were 0.69 and 1.17 T/ha, respectively (Table 4). The results indicate the success of breeding black soybeans for high yield when compared to Sukhothai 3, due to the RILs from all crosses showing a greater yield potential than the commercial Sukhothai 3 variety. The protein content of RILs and Sukhothai 3 during the dry season was higher than in the rainy season. During the rainy season, all RILs except SJ5xKKUSB–108–25–2–1 had more protein content than Sukhothai 3 (Table 4), whereas, during the dry season, KKU35xKKUSB–108–12–4–3, SJ5xKKUSB–108–30–3–7, NSW1xKKUSB–108–49–3–3, and NSW1xKKUSB–108–49–3–6 had more protein content than Sukhothai 3 (Table 4).

4. Discussion

The different colors of soybean grains, such as black, brown, green, yellow, and bicolor, are caused by the diverse biosynthesis of the pigments and accumulated pigment abundance in the seed coat belonging to the combination of multi-loci involved in the flavonoid-based pigmentation process [27]. This information demonstrates that genetic background and gene combination are involved in the seed coat color of soybeans. During population development, this phenomenon divests the seed coat color in the heterozygous population [42]. Although soybean seed color is a moderately complex trait controlled by multi-loci, only a few loci can be detected using a single bi-parental segregating population [38]. This study reveals that the black grain color of the soybean trait can be transferred from KKUSB-108 to all four commercial soybean varieties through pedigree methods (Figure 1). The F₂ population establishes the characteristics of the soybean seed coat, such as green, brown, yellow, and black. Generation advancement is achieved by selecting the soybean line with uniform black grains and then fixing the genotype of each RIL appropriated for the black soybean breeding program. In the F₃ and F₄ populations, soybean seed coats are black and brown. After the F₅ populations, the lines are inbred to create genetically stable recombinant lines. The results indicate that continued selection of the black seed coat genotype from the F₂ to F₅ populations through the pedigree and phenotypic performance can be successful in a black soybean breeding program (Figure 1).

Over the past 20 years, Thailand’s total soybean production area has drastically decreased from 0.30 million hectares in 2000 to 0.03 million hectares in 2020 [43]. The aging population of soybean farmers and the need for more sophisticated mechanical technologies in soybean cultivation are two constraints. For farmers, mechanized soybean harvesting is a dependable and economical choice. When utilizing a mechanical combine harvester, the FPH is a critical characteristic [34]. The FPH must be at least 12 cm high in soybeans to reduce seed yield loss from pods below the cutter level [35]. The FPH of RILs in this study ranged from 9.27 cm (CM60xKKUSB–108–64–4–8) to 15.02 cm. (KKU35xKKUSB-108-24-5-7) (Table 3). The first pod height of RILs from the KKU35 parental cross provided a high first pod height in both seasons because this cross was bred from two parents with a high pod height (FPH of KKU35 was up to 10.6 cm, and KKUSB-108 measured 14.7 cm). The continued selection of the black soybean genotype with FPH from the F₂ to F₅ populations through pedigree and phenotypic performance as demonstrated in this study can be successful in the black soybean breeding program (Figure 1 and Figure 2).

Soybean improvement programs emphasize various traits, including yield and yield components. As a result, modern soybean breeding lines have shown significantly higher yields than those in the past [44]. In this study, the yield and protein content of black soybean RILs were affected by season, genotype, and G x E interaction (Table 2). During the rainy season, the average yield was 1.44 T/ha, with a 34.75% protein content. In contrast, during the dry season, the average production was 1.66 T/ha, with a protein content of 41.22% (Table 4). Environmental variability significantly impacts soybean yields across various regions. This is primarily due to fluctuations in agricultural factors during the rainy and dry seasons, with weather conditions playing a particularly crucial role [45]. In this study, the RIL SJ5xKKUSB–108–25–2–1 consistently produced high yields during the rainy and dry seasons, unlike other RILs, whose yields varied by season (Table 4). The potential yield of black soybean RILs varies geographically due to differing climatic and environmental conditions [45,46]. Consequently, it is necessary to test the stability and adaptation of these lines across multiple locations. However, soybeans are considered a minor crop in Thailand. To effectively introduce improved black soybean RILs to specific areas, it is essential to integrate them with other cropping systems, such as rotation and intercropping [24].

Interestingly, the RILs developed in this study include black grain, high yield, high FPH, and early maturity. The NSW1xKKUSB-108-49-3-6 has the shortest harvest date of approximately 87 days in the rainy and dry seasons (Table 3). This RIL is suitable for growing as a rotational crop in a planting system, along with other major crops in Thailand, such as sugarcane, maize, and rice. The cropping system improves soil properties and soybean is an alternative crop that can increase farmers’ incomes. Their short growth duration allows soybean crops to rotate in a cropping system. The same strategy was used by Maranna et al. [47], who bred soybeans for early maturity to fulfill farmers’ demand in Central India for a soybean–potato–wheat/soybean–wheat cropping system. Soybeans are minor crops that are neglected for producing lower yields in Thailand. They usually compete with other crops in the same production area, especially corn. However, corn production requires more inputs, such as chemical fertilizers, herbicides, pesticides, and especially irrigation. Consequently, soybeans outperform corn in sustainable agriculture schemes because soybeans require less input than corn and are also beneficial for soil health. Based on the yield potential of black soybean RILs, the benefits of anthocyanins, high protein content, and short maturity have led to the suitability of black soybean RILs in agricultural production systems, potentially leading to Thailand initiating black soybean production in the future.

5. Conclusions

The black soybean RILs with high first pod height were successfully bred through crossbreeding the commercial yellow soybean varieties KKU35, SJ5, NSW1, and CM60 with the KKUSB–108 black soybean germplasm using the pedigree selection method. The black soybean RILs SJ5xKKUSB-108-25-2-1, SJ5xKKUSB-108-30-3-7, NSW1xKKUSB-108-49-3-6, and CM60xKKUSB-108-64-4-8 consistently showed high yields and greater FPHs across seasons. Additionally, NSW1xKKUSB-108-49-3-6 had a protein content of up to 44.21% in the dry season and shorter maturity, making it suitable for rotation cropping systems in areas with limited growing seasons. These results provide a valuable source of black soybean RILs with significant FPHs for Thailand’s future breeding programs. The influence of season, genotype, and their interaction on yield and yield components suggests that future trials in diverse environments are needed to evaluate these RILs further.