**3. Biotechnological approaches for improving the fatty acid composition**

#### **3.1 Transgenic soybeans with improved fatty acid profile**

Soybean is widely recognized as a dual-use crop because of its high protein and oil content [29], and several loci controlling both the traits have been identified. The negative correlation between these two traits [88] pose a challenge in genetic improvement programs. Introducing a transgene that can specifically modulate one pathway without disrupting the other can be useful to overcome the linkage between oil and protein. Several transgenic approaches have been tried to improve seed oil content in oilseed crops, In Arabidopsis, transcription factor gene, *WRI1,* and metabolic enzyme, acetyl-CoA carboxylase have been targeted [89, 90]. In soybean, Lardizabal et al. [91] first reported the development of a transgenic soy crop with increased oil that shows no major impact on protein content or yield. They achieved an increase in oil by 1.5% (by weight) in the mature seed by expressing a codon-optimized version of a diacylglycerol acyltransferase (DGAT)-2A from the soil fungus *Umbelopsis ramanniana* in soybean seed during development. Later, increased oil content of soybean seeds by an average of 3% was also reported with the use of an improved variant of soybean type 1 DGAT [92].

In recent years, RNA interference (RNAi) has gained significant attention due to its success for efficient metabolic engineering across the plant species. RNAi uses small interfering RNAs (siRNAs) to mediate the degradation of mRNA to regulate the expression of a desired plant gene. Using this approach, Flores et al. [93] showed that silencing of *GmFAD3* by siRNA caused a reduction in the ALA contents in *fad3*-mutant. A similar approach was used by Wagner et al. [94] for simultaneous suppression of soybean *FAD2* and *fatty acyl*-*ACP thioesterase (FATB)* genes to produce soybean seed with low-saturated, low-polyunsaturated oil phenotype.

Many studies in the recent past have demonstrated the role of *GmFAD2* family members in metabolically engineered oilseed plants. Using antisense RNA mediated posttranscriptional gene silencing approach, Zhang et al. [95] were successful in inhibiting the expression of *Gmfad2-1b* to develop transgenic soybean lines with increased oleic acid contents up to 51.7%. To simultaneously elevate stearic acid and reduce PUFA content in soybean, Park et al. [96] introduced the mangosteen (*Garcinia mangostana*) stearoyl-ACP thioesterase into soybean and subsequently stacked it with a soybean event that is down-regulated in both palmitoyl-ACP thioesterase activity and Δ12 fatty acid desaturase activity in a seed-specific fashion. This approach generated soybeans with a seed lipid phenotype of approximately 11–19% stearic acid and approximately 70% oleic acid. Recently, the introduction of the *PfFAD3-1* gene from Lesquerella (*Physaria fendleri*) into soybean resulted in an increase in the ALA content up to 42% in the seeds of T2 homozygous plants [97].

It is important to note that the transgenes expressing RNAi constructs are subject to variation in transgene expression, and hence a large number of events need to be screened to select the candidate providing stable expression. They also need to go through the regulation process, which is not only expensive but also timeconsuming. Nevertheless, these approaches are expected to guide further improvement in the fatty acid composition without largely affecting the other traits, mainly the protein content and yield.

### **3.2 Targeted mutagenesis to improve ω-3 fatty acid contents**

Targeted genome engineering (also known as genome editing) using designed nucleases has emerged as an alternative to conventional plant breeding and transgenic means to improve crop plants [98]. The discovery of sequence-specific nucleases (SSNs) such as TAL effector nucleases (TALENs) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9, made it possible to introduce targeted knockout mutations within gene/s of interest [99, 100]. These SSNs make DNA double-stranded breaks at defined genomic loci, which are subsequently repaired by two main DNA repair pathways, which result in frameshift mutations that often create genetic knockouts. Such knockout lines have been generated across the plant species, making genome editing an emerging tool for trait improvement.

Using genome editing approach, Haun et al. [101] engineered TALENs to recognize and cleave conserved DNA sequences in *FAD2–1A* and *FAD2–1B* genes. In the

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

plants that carried homozygous mutations in both *FAD2–1A* and *FAD2–1B* genes, oleic acid was increased from 20% to 80% and linoleic acid was reduced from 50% to 4%. Further reduction in the linoleic acid (up to 2.5%) was achieved by stacking mutations within *FAD2–1A* and *FAD2–1B* with mutations in *FAD3A* [44]. Such an approach of TALENs-mediated targeted mutagenesis of *FAD2* was found to be effective in the development of the high oleic peanut (*Arachis hypogaea* L.) varieties [102]. The low to average mutagenic frequency by TALENs has been observed in the genome editing studies done so far in peanut and soybeans. The efficiency of genome editing can further be enhanced by using the CRISPR/Cas system.

In recent years, the CRISPR/Cas9 system has revolutionized functional genomics due to its simplicity, efficiency, cost-effectiveness, and versatility [103]. The CRISPR system has two components: a nuclear-localized CRISPR-associated (Cas) 9 protein and a guide RNA (gRNA). Cas9 is a large protein containing two nuclease domains, whereas the gRNA is a synthetic 100 nucleotide RNA molecule, of which the first ~20 nucleotides are the targeting site, and the 3′ end forms a hairpin structure that interacts with the Cas9 protein [104]. The Cas9 and the gRNA interact to identify DNA sequences complementary to the gRNA and generate a DNA doublestrand break, which, after a repair result in genomic insertion or deletion (indel) mutations.

In plants, the CRISPR-Cas9 system has been effectively used in many species such as *Arabidopsis thaliana*, *Nicotiana benthamiana*, rice, tobacco, sorghum, wheat, and maize [105]. In soybean, CRISPR/Cas9-mediated genome editing has been successful in targeting DNA mutations in genes for soybean hairy roots and flowering [106–109], plant architecture and yield [110], plant height [111], and seed storage protein genes [112]. However, researchers have started using this system to improve fatty acid composition. Do et al. [113], designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of *GmFAD2–1A* and *GmFAD2–1B* to yield a high-oleic, low-linoleic, and low-ALA phenotype in soybean. In this study, dramatic increases in oleic acid content to >80%, and decreases of 1.3–1.7% in linoleic acid were observed in the T1 seeds derived from CRISPR-edited plants homozygous for both *GmFAD2* genes. In a similar study, increases in oleic acid from 17.10% to 73.50%, and decreases in the linoleic acid content from 62.91% to 12.23% have been reported by inserting mutations in *GmFAD2–1A* and *GmFAD2–2A* soybean fatty acid desaturase mutants based on CRISPR/Cas9 Technology [114]. Overall, these studies demonstrated the CRISPR/Cas9 system as a rapid and highly efficient method to simultaneously edit homeologous soybean genes to facilitate gene discovery and breeding programs.
