**2.3 Increasing ω-3 fatty acid content for improving ω-6/ω-3 ratio in soybean**

Soybean production focuses on providing high protein meals for livestock and the manufacture of vegetable oils in both Western and Asian countries, while soybean has traditionally been used as a staple food in many Asian countries [2, 74]. The consumption of soy foods has been increasing in North America, following the recognition of the health benefits of soy foods.

Since the shortage of resources in cultivated soybean with elevated ALA content [75], researchers tried to find suitable genetic resources to develop new cultivars with high ALA concentrations in soybean breeding programs. Wild soybean can be a possible resource to achieve the goal to increase ALA concentration because those soybeans have an average of 15% ALA concentration, which is almost twice the ALA concentration present in the cultivated soybean [76]. Cultivated soybeans have an ω-6/ ω-3 ratio of 6–7:1, whereas wild soybeans have an ω-6/ ω-3 ratio of 3–4:1, which has better health benefits [76–78]. Thus, wild soybean can be exploited as a genetic resource to develop soybean lines with high ALA concentrations, although exploiting wild soybeans in breeding programs is challenging due to their poor agronomic traits. Several studies reported soybean lines with elevated ALA from wild soybean using conventional breeding methods. Asekova et al. [77] reported that three recombinant inbred lines with elevated ALA concentrations from an interspecific cross between *G. max* and *G. soja* were stable for the accumulation of ALA across the environments. Also, since *G. soja* as donor plant was backcrossed with three different cultivars, new genotypes with elevated ALA concentration and agronomically similar to the cultivated soybeans have been developed [78]. These soybean lines developed by classical breeding could be exploited as genetic resources for the development of novel soybean cultivars with high levels of ALA concentration, which could be sources of ω-3 fatty acids.

To date, there have been few genetic mapping studies with high ALA concentration in soybean. Shibata et al. [79] identified four QTLs controlling ALA concentration in the wild soybean accession Hidaka 4. Also, Ha et al. [80] identified nine putative QTLs controlling ALA concentration in a wild soybean accession PI 483463. According to these studies, high ALA concentrations in wild soybean were controlled by multiple QTLs. Besides, Pantalone et al. [81] suggested that high ALA concentration in wild soybean was controlled by a different set of desaturase alleles from cultivated soybean. Recently, the application of gamma-ray irradiation has generated new mutant soybeans with a high level of ALA concentration [82]. They concluded that the phenotype of high ALA concentration in these mutant lines was related to *FAD3* gene expression levels, although they observed no direct relationship between elevated gene expression level and gene sequence variations. Taken together, we assume that increased expression levels of *FAD3* genes during seed development may be associated with the gene expression regulators.

Since *FAD2* genes play an important role in regulating ω-6/ω-3 ratio in soybean, *FAD2* mutant alleles were found to increase in oleic acid and decrease in linoleic acid contents [40, 83, 84]. Populations segregating for *FAD2–1A* and *FAD2–1B*

*Breeding Strategy for Improvement of Omega-3 Fatty Acid through Conventional Breeding… DOI: http://dx.doi.org/10.5772/intechopen.95069*

#### **Figure 3.**

*Schematic representation of the genetic improvements in the steps that result in significant reductions in* ω*-6/*ω*-3 ratio in soybean. The mutant alleles of FAD2-1A or FAD2-1B increase the oleic and reduce linoleic acid content, whereas, various alleles of FAD3 cause increases in the* α*-linolenic acid contents. These improvements significantly alter the* ω*-6/*ω*-3 ratio in soybean seed oil.*

mutant alleles have been investigated for increases in oleic acid content [40]. By combining either of a mutant allele in M23 (the deletion of the *FAD2–1A* gene) or 17D (*FAD2–1A* S117N) with either of a missense mutant allele in *FAD2-1B* from PI 283327 (*FAD2-1B* P137R) or PI 567189A (*FAD2-1B* I143T), soybean genotypes with 77.3–82.2% oleic acid content were developed [84]. The ω-6/ω-3 ratio in these lines ranged from 0.6 to 1.3. Similarly, progenies containing *FAD2-1A* allele from the 17D lines, and *FAD2-1B* allele from S08-14788 were found to show an ω-6/ ω-3 ratio in the range of 0.62-0.97 [85]. These soybean genotypes had high oleic acid content (~80%) and lower ω-6/ω-3 ratio, but the overall ω-3 acid content (~5%) was also very low [86]. Kulkarni et al. [87] suggested the genetic improvement of the system to increase ALA concentration with a balanced ω-6/ω-3 ratio. Soybean containing either of the *FAD2-1* mutant alleles with ALA-related alleles from wild soybean reduced the seed ω-6/ω-3 ratio as well as increased ω-3 fatty acid concentration. Among *FAD2* genes, soybean genotypes with a mutant allele of the *FAD2-1A* gene had higher oleic acid and ALA content in soybean oil than one with *FAD2-1B* mutant allele. Further genetic improvements in the FA biosynthetic pathways were made by combining mutant alleles of either of *FAD2-1A* or *FAD2-1B* genes with alleles governing ALA in wild soybeans to develop soybean genotype with lower ω-6 and higher ω-3, resulting in low ω-6/ ω-3 ratio (**Figure 3**; [87]). Similar genetic improvements involving new sources of ALA-controlling alleles from the wild soybeans can guide development of soybeans with a balanced ω-6/ω-3 ratio in their seed oils.
