Soybean [Glycine max (L.) Merr.] seeds contain protein, carbohydrate, oil, vitamins, minerals, and many functional components. Also, there are antinutritional factors and several allergenic proteins in the raw mature soybean. Kunitz trypsin inhibitor (KTI) protein of mature soybean seed is a main antinutritional factor in soybean seed. KTI protein is a small and non-glycosylated protein containing 181 amino acid residues with 21.5 kDa. KTI protein was first isolated and crystallized from soybean seeds by Kunitz (1945). Kunitz trypsin inhibitor protein strongly inhibits trypsin, thus reducing food intake by diminishing digestion and absorption. Five electrophoretic forms of KTI have been discovered. The genetic control of four forms, Ti^a, Ti^b, Ti^c, and Ti^d, has been reported as a codominant multiple allelic series at a single locus (Sing et al. 1969; Hymowitz and Hadley 1972; Orf and Hymowitz 1979). Orf and Hymowitz (1979) found that the fifth form does not exhibit a soybean trypsin inhibitor-A2 band and is inherited as a recessive allele designated ti. Orf and Hymowitz (1979) identified two soybean accessions (PI157440 and PI196168) which lacks KTI protein from USDA germplasm collection. The Ti locus has been located on linkage group 9 in the classical linkage map of soybean (Hildebrand et al. 1980; Kiang 1987), which was integrated in linkage group A2 (chromosome 8) of the USDA/Iowa State University soybean molecular linkage map (Cregan et al. 1999).

β-conglycinin (7S globulin) and glycinin (11S globulin) are the major components of storage protein in soybean. β-conglycinin consists of three subunits, α′, α, β (Thanh and Shibasaki 1976) and exhibits poorer nutritional and food processing properties than glycinin. Also, β-conglycinin contains much less sulfur-containing amino acid, methionine and cysteine, than glycinin (Koshiyama 1968). Several mutant lines affecting accumulation of β-conglycinin have been identified in soybean germplasms. Kitamura and Kaizuma (1981) identified Keburi, which was characterized by the absence of the α′-subunit of β-conglycinin. Kitamura et al. (1984) reported that the absence of α′-subunit were controlled by single recessive alleles, cgy₁. Wild soybean line, QT2, which lacks α′-subunit was also identified. In the QT2 line, the deficiency of α′-subunit is controlled by a single dominant gene, Scg (Hajika et al. 1998). Scg gene was inherited independently with cgy₁ gene. Studies also showed that absence of the α′-subunit in QT2 was inherited independently from the presence of lipoxygenase isozymes. Hayashi et al. (2000) identified AFLP marker to be tightly linked to the gene for deficiency of the β-conglycinin. Highly negative correlation between the contents of β-conglycinin and glycinin was reported by Ogawa et al. (1989). Breeding of soybean containing large amount of glycinin compared with current varieties is possible by selecting strain of soybean that does not exhibit or lacks α′,α, and β-subunits of β-conglycinin. KTI and α′protein limit the utilization of raw soybeans as direct feed requiring a heating step to denature the activity and energy costs as well as altering the physical properties of the soybean proteins. Also excessive heat treatment may lower amino acid availability. The genetic elimination of these factors could be an alternative to the heat treatment. Development of of new soybean cultivar with both KTI and α′-subunit protein free is needed to improve the nutrition values and to modify the food processing properties of soybeans. The objective of this research was to develop a new soybean strains with KTI and α′-subunit protein free (titicgy₁cgy₁ genotype) and proper agronomic traits.

Breeding population

Two parents were used to develop breeding population. BL-1 parent has Cgy₁ gene (α′-subunit of β-conglycinin protein present, Cgy₁Cgy₁ genotype) and ti gene (KTI protein absent, titi genotype). 15G1 parent has cgy₁ gene (α′-subunit of β-conglycinin protein absent, cgy₁cgy₁ genotype) and Ti gene (KTI protein present, TiTi genotype). Seed traits and genotypes for Cgy1(cgy1) and Ti(ti) alleles of two parents are presented in Table 1. The seeds of BL-1 and 15G1 parents were planted to cross in a greenhouse. The crosses of BL-1 (Cgy₁Cgy₁titi) x 15G1 (cgy₁cgy₁TiTi) were made and F₁ seeds were obtained. F₁ seeds obtained were planted in greenhouse. F₁ hybridity was confirmed on morphological traits. F₂ seeds from F₁ plant confirmed were harvested. The F₂ seeds were analyzed to screen the seed with cgy₁cgy₁titi genotype (KTI and α′-subunit protein free).

Table 1 . Seed coat color, hilum color and genotype for Cgy1 (cgy1) and Ti (ti) alleles of the two parents used in this experiment.

Parents	Trait/genotype
	Seed coat color	Hilum color	KTI protein	Ti(ti)	α′-subunit protein	Cgy1 (cgy1)
BL-1	Yellow	Yellow	Absent	titi	Present	Cgy1Cgy1
15G1	Yellow	Yellow	Present	TiTi	Absent	cgy1cgy1

Determination of α′-subunit protein by SDS-PAGE

Crude protein from parent and each F₂ seeds extracted to determine the presence or absence of α′-subunit protein of β-conglycinin electrophoretically. A piece of cotyledon from parent and each F₂ seed was removed and was incubated for 30 min (at room temperature) in 1 ml Tris-HCl, pH8.0, containing 1.56% v/v β- mercaptoethanol. After centrifugation, 50 μlof the supernatant was added to an equivalent amount of 5X sample buffer [10% w/v sodium dodecyl sulfate (SDS), 50% v/v glycerol, 1.96% v/v β-mercaptoethanol, 1M Tris-HCl, pH 6.8]. The samples were boiled at 97°C for 5 min and then centrifuged. Two microliters of the supernatant were loaded on a 12% acrylamide SDS polyacrylamide gel electrophoresis (SDS-PAGE) medium gels in Owl Separation Systems Inc (Model:P9DS, Portsmouth, NH, USA). Electrophoresis was performed at 120 V for 7 hrs. Gels were stained overnight in an aqueous solution of 0.25 g Coomassie blue R250, 10% acetic acid, and 45% methanol. The gels were then destained with destaining solution (5% acetic acid, 14% methanol) for several hours. A Wide-Range SDS-PAGE molecular mass standard (Sigma Marker™, Product Code: M4038, St.Louis MO, USA) containing the 72kDa (for α′-subunit) was used to aid recognition of samples lacking the α′-subunit of β-conglycinin protein.

Determination of KTI protein by western blot analysis

Proteins of parent and each F₂ seed were separated by 10% or 12% SDS-PAGE, and transferred onto Immobilon- Pmembrane (PVDF, Millipore). After blocking for 2hr in TBS buffer [20mM Tris(pH7.5), 150mM NaCl, and 0.1% Tween20] with 5% nonfat dried milk (Carnation, Glendale, CA) at room temperature, the membrane were incubated with KTI antibody for 1hr. After washing in TBS buffer three times, the blot was incubated with a horseradish peroxidase conjugated secondary antibody, and the complex was visualized using an enhanced chemiluminescence kit (Amersham, Bucking-hamshire, UK). The thickness of band was then determined visually.

Selection of titicgy₁cgy₁ genotype (KTI and α′-subunit protein free)

The F₂ seeds with titicgy₁cgy₁ genotype (KTI and α′-subunit protein free) were planted to advance F₂ plant generation. F₂ plants with a proper agronomic traits were individually harvested. Two strains with a proper agronomical traits were advanced to F₄ plant generation. Random F₅ seeds from each F₄ plant harvested were used to confirm absence for both KTI and α′-subunit proteins. Plant height and 100-seed weight was recorded on the F₅ plant generation. Mean values of plant height and 100-seed weight were compared by Duncan’s multiple range test at the 5% level. Scheme for development of new soybean strain with titicgy₁cgy₁ genotype is presented in Figure 1.

Figure 1.

Scheme for development of new soybean strains with titicgy₁cgy₁ genotype (KTI and α′-subunit protein free)

A total 168 F₂ seeds from the cross of BL-1 and 15G1 parents were obtained and analyzed for the segregation of Kunitz trypsin inhibitor (KTI) protein and α′-subunit protein of β-conglycinin. A part of the SDS-PAGE pattern for α′- subunit protein that appeared in the parents and F₂ seed generation is shown in Figure 2.

Figure 2.

Segregation of α′-subunit protein in the parents and F₂ seeds. Arrow is the α′-subunit protein band. S: marker, P1: BL-1, P2: 15G1. +, -: presence and absence of α′-subunit protein, respectively

Bands for α′-subunit of β-conglycinin were segregated in F₂ seeds. Segregation pattern for Kunitz trypsin inhibitor (KTI) protein that appeared in the parents and F₂ seed generation is shown in Figure 3.

Figure 3.

Segregation of the Kunitz trypsin inhibitor (KTI) protein in the parents and F₂ seeds. Arrow is the KTI protein band. P1: BL-1, P2: 15G1. +, -: presence and absence of the KTI protein, respectively

The segregation data for KTI protein and α′-subunit protein of β-conglycinin protein in the F₂ seed generation are presented in Table 2.

Table 2 . Segregation for the presence or absence of Kunitz trypsin inhibitor (KTI) and α′-subunit proteins in F2 seed generation.

Seed protein		Seed number		χ2value (9:3:3:1)	P
KTI	α′-subunit	Observed	Expected
+	+	104	94.5	5.12	0.5 - 0.1
+	-	30	31.5
-	+	21	31.5
-	-	13	10.5

+, -: presence and absence of protein..

The segregation ratios of 9 : 3 : 3 : 1 (104 Ti_Cgy₁_: 30 Ti_cgy₁cgy₁: 21 cgy₁cgy₁Ti_: 13 titicgy₁cgy₁) between Ti gene and Cgy₁ gene in F₂ seeds were observed (χ²= 5.12, P = 0.5-0.10). This data showed that Ti gene was inherited independently with the Cgy₁ gene in soybean. These results supported the previous reports (Kitamura et al. 1984; Orf and Hymowitz 1979; Sung et al. 2010). Only thirteen F₂ seeds with recessive allele titicgy₁cgy₁ genotype were planted and Ten F₂ plants were individually harvested. Agronomic traits such as plant type, plant height, maturity date, lodging, seed quality, and 100-seed weight were evaluated on the ten F₃ plant strains with titicgy₁cgy₁ genotype. Two F₄ plant strains with a proper agronomical traits were selected and harvested. Random F₅ seeds from each F₄ plant strain were used to confirm absence for both KTI and α′-subunit proteins (Fig. 4).

Figure 4.

Confirmation of the α′-subunit protein free (Ⓐ) and Kunitz trypsin inhibitor (KTI) protein free (Ⓑ) in the parents and new strains. Arrows are the α′ -subunit band (72 kDa) and KTI protein band (21.5 kDa). P1: BL-1, P2: 15G1, S1, S2: new strain (titicgy₁cgy₁ genotype). +, -: presence and absence of α′- subunit and KTI proteins, respectively

Kunitz trypsin inhibitor (KTI) and α′-subunit proteins were not observed in the mature F₅ seed of two new strains. Agronomic traits of two parents and two new strains with titicgy₁cgy₁ genotype are presented in Table 3.

Table 3 . Agronomic traits of parents and two new strains selected in this experiment.

Parent/ strain	Plant height (cm)	Seed weight (g/100 seed)	Seed coat color	Hilum color
BL-1	63az	24.5a	Yellow	Yellow
15G1	75b	32.1b	Yellow	Yellow
S1	65a	29.2b	Yellow	Yellow
S2	66a	26.1a	Yellow	Yellow

^zDifferent letters in the column are significantly different by DMRT at 5%.

Two strains (S1 and S2) have yellow seed coat and hilum. Plant height of S1 strain was 65 cm compared to the parents of 63 ~ 75 cm. The 100-seed weight of S1 strain was 29.2 g compared to the parents of 24.5 ~ 32.1 g. Plant height of S2 strain was 66 cm compared to the parents of 63 ~ 75 cm. The 100-seed weight of S1 strain was 26.1 g compared to the parents of 24.5 ~ 32.1 g. Plants and seeds of two new strains with titicgy₁cgy₁ genotype are shown in Figure 5.

Figure 5.

Plants (up) and seeds (down) of the two new strains (S1 and S2) with titicgy₁cgy₁ genotype

Two strains selected in this research will be used to improve new cultivar with free of KTI and α′-subunit proteins.