Soybean [Glycine max (L.) Merr.] seeds contain high quality proteins, essential fatty acids, vitamins, fiber, and phytochemicals. However, antinutritional and harmful immunological components such as lipoxygenase protein, Kunitz trypsin inhibitor (KTI) protein, 7S α'-subunit protein, and stachyose component also exist in mature soybean seeds. Lipoxygenase protein causes undesirable grassy and beany flavors in foods containing soybean due to the oxidation of polyunsaturated fatty acids and three lipoxygenases (Lox1, Lox2 and Lox3) that exist in mature seeds. Kunitz trypsin inhibitor (KTI) protein, which was isolated and crystallized by Kunitz (1945) is a small non-glycosylated protein possessing 181 amino acid residues with 21.5 kDa. KTI protein severely inhibits trypsin, thus reducing food intake by diminishing digestion and absorption. Two major components of storage protein in soybean are β-conglycinin (7S globulin) and glycinin (11S globulin). The α, α′, and β subunits are major constituents of β-conglycinin. They exhibit poorer nutritional and food processing properties than glycinine (Thanh & Shibasaki 1976). Also, methionine and cysteine, which contain sulfur, have much less β-conglycinin than glycinin (Koshiyama 1968). About 11-15% of total carbohydrates in soybean seed are sucrose, stachyose and raffinose. Raffinose and stachyose are considered antinutritional factors because humans cannot digest them after absorption.

Because of these antinutritional factors and allergens that exist in raw soybean seeds, heat treatment or other methods are needed to eliminate or reduce these components and secure the efficiency of nutrient absorption and food safety. But these treatments cause some changes and reducs soybean quality (Chen et al. 2019). Also, heat inactivation of the lipoxygenase at an industrial level not only incurs extra cost but also affects the solubility and functionality of proteins (Macleod & Ames 1988). Even after heat treatment and fermentation, antigenic proteins remain in soybean food (Wilson et al. 2008). Genetic removal or reducing of antinutritional and allergenic factors such as lipoxygenase, KTI, 7S α’ subunit protein and stachyose that exist in mature soybean seeds is needed in the soybean food industry. So far, ‘Jinpumkong’ and ‘Chosen’ cultivars that are lipoxygenase-2,3 protein free and ‘Jinpum#2’, ‘Miso’, and ‘Jinyang’ cultivars that are lipoxygenase-1,2,3 protein free have been released (Kim et al. 1996, Kim et al. 1997, Chung 2014, Kim et al. 2015, Chung 2017). Also, ‘Gaechuck#1’ and ‘Gaechuck#2’ cultivars with an absence of lipoxygenase-2,3 protein and KTI protein have been released (Chung 2009a, Chung 2009b). A soybean cultivar with large yellow seeds that is free of lipoxygenase, KTI, and 7S α’ subunit proteins and has low stachyose content was developed.

Breeding process

An intermediate parent (F4-4) and breeding line (15G1) were used to improve new lines with traits of yellow seed coat, large seed size, and free of lipoxygenase, Kunitz trypsin inhibitor (KTI), and 7S α' subunit proteins and low stachyose content. F4-4 used as a female has traits of yellow seed coat, medium seed size (25.0 g/100 seed), and free of lipoxygenase and 7S α' subunit proteins. 15G1 used as a male parent has a brown seed coat, large seed size (34.5 g/100 seed), free of lipoxygenase and KTI proteins, and low stachyose content. F₁ seeds from the cross were obtained and planted in a greenhouse in 2009.

The presence or absence of 7S α' subunit protein and KTI protein in each of the F₂ seed was tested by SDS- PAGE and western blot analysis, respectively. F₂ seeds were planted in May 2010. Single F₂ plants were harvested based on plant type, height and maturity. Random F₃ seeds of each F₂ plant harvested were analyzed to select strains with low stachyose content (rs2rs2 genotype) by HPLC. Thirty lines free of 7S α' subunit and KTI proteins and with low stachyose content were planted and harvested in 2011. The pedigree selection method was used through F₅ and F₇ generation to select plants with a yellow seed coat, large seed size, free of lipoxygenase, KTI, and 7S α' subunit proteins and low stachyose content. One F₈ line with acceptable agronomical traits such as flowering date, maturity date, growth habit and lodging was selected in 2015. Qualitative and quantitative traits were recorded in 2018 at a location in Jinju. Agronomic traits such as lodging, shattering and reaction to disease were recorded according to the standard soybean cultivation manual of the RDA, Korea. Yield, protein and oil content, and insect resistance were tested by the Korea Seed & Variety Service. Genealogical diagram and selection procedures are shown in Fig. 1 and Fig. 2.

Fig. 1. Genealogical diagram of ‘Hayoung’.

Fig. 2. Pedigree diagram of ‘Hayoung’.

Yield trials and agronomic traits

The breeding line was yield tested in a preliminary yield trial in 2015 and advanced yield trial in 2016 at a research farm of GNU. The planting date was June 10. A regional yield trial was conducted in 2018 at four locations, and the planting date was June 15. The experimental design for the yield trial was RCBD with three replications. Plots consisted of three rows. The test area was 7.2 ㎡ and planting distance was 60x 15 cm, with two plants per each. Ten plants from the center row of each plot were used for agronomic traits and harvested for yield. The flowering date was measured as the date when 50% of the plants had at least one flower. Plant height was measured in cm from the soil surface to the tip of the main stem at maturity. Lodging was visually rated at maturity using a scale from 1 to 9, where 1=almost all plants erect, and 9=almost all plants down. Shattering was visually rated at maturity using a scale from 1 to9, where 1=almost all pods not shattered, and 9=almost all pods shattered. The maturity date was measured as the date when almost all pods matured. Seed weight was measured from 100 randomly chosen seeds air-dried to a seed moisture content of about 15% after harvesting. Other agricultural traits were investigated according to the Korea Seed & Variety Service, Survey and Analysis Standard.

Confirmation of lipoxygenase and 7S α' subunit proteins by SDS-PAGE

Crude protein from the random seed of the check cultivar (‘Daewon’) and ‘Hayoung’ was obtained to identify the presence (‘+’) or absence (‘−’) of lipoxygenase and 7S α' subunit proteins. Part of the cotyledon from random seeds was removed and incubated for 30 min in 1 ml of Tris-HCl, pH 8.0, and 1.56% v/v β-mercaptoethanol. About 50 μl of the supernatant through centrifugation was added to an equivalent amount of 5X sample buffer containing 1 M Tris-HCl, pH 6.8, 50% v/v glycerol, 1.96% v/v β-mercaptoethanol, and 10% w/v sodium dodecyl sulfate (SDS). The sample obtained was boiled at 97°C for 5 min and centrifuged. About 2 μl of the supernatant was loaded on a 12% acrylamide SDS polyacrylamide gel electrophoresis medium gel from Owl Separation Systems, Inc (Model: P9DS, Portsmouth, NH USA). After electrophoresis at 120 V for 7 h, gels were stained. The gels were destained in destaining solution for several hours. The presence or absence of lipoxygenase protein (97 kDa) and 7S α' subunit protein (72 kDa) was confirmed.

Confirmation of KTI protein by western blot analysis

Proteins obtained from the seed of the check cultivar (‘Daewon’) and ‘Hayoung’ were separated by 12% SDS-PAGE, and transferred onto an Immobilon-P membrane (PVDF, Millipore). After blocking for 2 h in TBS buffer containing 0.1% Tween 20, 20 mM Tris (pH 7.5), 150 mM NaCl, and 5% nonfat dried milk (Carnation, Glendale, CA), the membrane were incubated with the antibody of KTI and lectin protein for 1 h. The blot was incubated with a horseradish peroxidase conjugated secondary antibody after washing in TBS buffer. Using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK), the complex was visualized. The presence or absence of KTI protein was determined visually.

Analysis of crude protein, oil and stachyose

The content of crude protein and oil for ‘Hayoung’ and check cultivar (‘Daewon’) was analyzed by the Soxhlet method and semimicro-Kjeldahl method, respectively. Stachyose content for ‘Hayoung’ and ‘Daewon’ cultivars was detected by HPLC (Sung et al. 2014).

Disease performance

Disease performance was conducted at a research field of Gyeongsangnam-do Agricultural Research & Extension Services in 2018. The ‘Daewon’ cultivar was used as a check cultivar. Twenty plants were used for the disease test. Necrosis and mosaic virus symptoms were investigated at V2 stage. Black root rot and bacterial pustule symptoms were investigated at R1 stage. Response of cercospora leaf spot and blight and symptoms of pod and stem blight were checked at R8 stage. Disease reaction was visually rated using a scale from 1 to 9 according to the Agricultural Science and Technology Research and Analysis Criteria (2012, Rural Development Administration).

Agronomic performance

Qualitative traits of ‘Hayoung’ are presented in Table 1. ‘Hayoung’ has purple flowers, tawny pubescence, a determinate growth habit, oval leaflet shape, and light yellow pods at maturity. The seed of ‘Hayoung’ has a yellow hilum and yellow seed coat color. The cotyledon color of the mature seed is yellow.

Table 1

Qualitative characteristics of ‘Hayoung’.

Cultivar	Growth habit	Flower color	Leaf shape	Pubescence color	Seed coat color	Hilum color	Cotyledon color
Hayoung	Determinate	Purple	Oval	Tawny	Yellow	Yellow	Yellow
Daewon	Determinate	Purple	Oval	Tawny	Yellow	Yellow	Yellow

Quantitative traits of ‘Hayoung’ are shown in Table 2. ‘Hayoung’ flowered on August 4, which is 5 days later than ‘Daewon’. ‘Hayoung’ matured on October 22, which is 2 days earlier than ‘Daewon’. The plant height of ‘Hayoung’ was 56 cm compared to the check cultivar (‘Daewon’) with 46 cm. The lodging score of ‘Hayoung’ was 0. The 100-seed weight of ‘Hayoung’ was 32.3 g larger than that of ‘Daewon’ (29.0 g). The stachyose content of ‘Hayoung’ was 3.1 g/kg, which was much lower than 12.7 g/kg of the check cultivar, ‘Daewon’. ‘Hayoung’ had a shattering score of 2.0 versus ‘Daewon’ with a score of 1.0.

Table 2

Quantitative characteristics of ‘Hayoung’ determined by regional yield trial in 2018

Cultivar	Flowering date	Maturity date	Plant height (cm)	Lodgingz (0-9)	100-seed weight (g)	Stachyose (g/kg)	Shatteringy (0-9)
Hayoung	August 4	Oct. 22	56±3.5ax	0	32.3±1.5ax	3.1±0.2ax	2.0
Daewon	July 31	Oct. 24	46±2.4b	0	29.0±1.4b	12.7±0.7b	1.0

^z0: not lodged, 9: completely lodged.

^y0: no shattering, 9: completely shattering.

^xMean±Standard Error, different letters in the column are significantly different by DMRT at 5%.

The yield of ‘Hayoung’ is shown in Table 3. The average yield of ‘Hayoung’ was 3.12 (Ton/ha) in the regional yield trials of 2018 at four locations.

Table 3

Yield of ‘Hayoung’ in the regional yield trials at four locations in 2018

Location	Hayoung (Ton/ha)	Daewon (Ton/ha)
Jinju	3.30	3.37
Sachun	3.24	3.32
Miryang	3.02	3.13
Muan	2.92	3.03
Mean	3.12ax	3.21az

^zSame letter is not significantly different at 5%

Traits for seed components of ‘Hayoung’ are shown in Table 4. Average seed protein and oil content on a dry weight basis were 37.9% and 16.0%, respectively, compared to 36.7% and 18.7% of the check cultivar, ‘Daewon’.

Table 4

Characteristics for seed components of ‘Hayoung’ estimated in 2018 at Jinju

Cultivar	Crude protein (%)	Crude oil (%)	Lipoxygenase	KTI	7S α' subunit
Hayoung	37.9	16.0	Absent	Absent	Absent
Daewon	36.7	18.7	Present	Present	Present

Random seeds of ‘Hayoung’ and ‘Daewong’ were used to confirm the absence of lipoxygenase, KTI, and 7S α' subunit proteins (Fig. 3).

Fig. 3. Confirmation of lipoxygenase (a), 7S α' subunit (b) and KTI (c) proteins in mature seed of ‘Daewon’ (C) and ‘Hayoung’ (H). +, -: presence (+) and absence (-) of lipoxygenase, 7S α' subunit and KTI proteins, respectively.

The check cultivar ‘Daewon’ contained lipoxygenase, 7S α' subunit and KTI proteins in the mature seed. However, lipoxygenase, 7S α' subunit and KTI proteins were absent in the mature seed of ‘Hayoung’.

Plants and seeds of ‘Hayoung’ are shown in Fig. 4.

Fig. 4. Phenotype of ‘Hayoung’ cultivar. (A): appearance of V6 stage in field, (B): appearance of R8 stage in field, (C): harvested seeds.

Disease Resistance

The reaction of ‘Hayoung’ to soybean diseases is presented in Table 5. ‘Hayoung’ was resistant to soybean necrosis, similar to the check cultivar (0 score). Also, both cultivars, ‘Hayoung’ and ‘Daewon’, were resistant to soybean mosaic. ‘Hayoung’ was resistant to Cercospora leaf spot and blight, black root rot, pod and stem blight. The resistance to bacterial pustule in ‘Hayoung’ was higher than ‘Daewon’ with scores of 0 and 1, respectively.

Table 5

Resistant degree of ‘Hayoung’ to soybean disease estimated in 2018

Cultivar	Necrosis (0-9)z	Mosaic virus (0-9)	Cercospora leaf spot and blight (0-9)	Black root rot (0-9)	Pod and stem blight (0-9)	Bacterial pustule (0-9)
Hayoung	0	0	1	0	1	0
Daewon	0	0	1	0	1	1

^z0: Resistant, 9:susceptible.

The soybean cultivar ‘Hayoung’, with large yellow seeds, free of lipoxygenase, Kunitz trypsin inhibitor (KTI), and 7S α'-subunit proteins and that has low stachyose content, was developed. ‘Hayoung’ was selected from the population derived from the cross between an intermediate parent (F4-4) and breeding line (15G1). ‘Hayoung’ has purple flowers, tawny pubescence, a determinate growth habit, and light yellow pods at maturity. The seed of ‘Hayoung’ has a yellow hilum and yellow seed coat color. Seed protein and oil content on a dry weight basis were 37.9 and 16.0%, respectively. ‘Hayoung’ flowered on August 4, which is 5 days later than ‘Daewon’. ‘Hayoung’ matured on October 22, which is 2 days earlier than ‘Daewon’. The plant height of ‘Hayoung’ was 56 cm compared to the check cultivar (‘Daewon’) with 46 cm. The lodging score of ‘Hayoung’ was 0. The 100-seed weight of ‘Hayoung’ was 32.3 g larger than that of ‘Daewon’ (29.0 g). The stachyose content of ‘Hayoung’ was 3.1 g/kg, which was much lower than 12.7 g/kg of the check cultivar, ‘Daewon’. ‘Hayoung’ had a shattering score of 2.0 versus ‘Daewon’ with a score of 1.0. The average yield of ‘Hayoung’ was 3.12 ton/ha in 2018. ‘Hayoung’ showed resistance to soybean necrosis, soybean mosaic virus, Cercospora leaf spot and blight, black root rot, pod and stem blight, and bacterial pustule. ‘Hayoung’ has been registered as a soybean cultivar (registration number: 8625, registration date: June 08, 2021) by the Korea Seed & Variety Service, Republic of Korea.