Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1

Abstract

Soybean is a major crop that is used as a source of vegetable oil for human use. To develop transgenic soybean with high a-linolenic acid (ALA; 18:3) content, the FAD3-1 gene isolated from lesquerella (Physaria fendleri) was used to construct vectors with two different seed-specific promoters, soybean b-conglycinin (Pb-con) and kidney bean phaseolin (Pphas), and one constitutive cauliflower mosaic virus 35S promoter (P35S). The corresponding vectors were used for Agrobacterium-mediated transformation of imbibed mature half seeds. The transformation efficiency was approximately 2%, 1%, and 3% and 21, 7, and 17 transgenic plants were produced, respectively. T-DNA insertion and expression of the transgene were confirmed from most of the transgenic plants by polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), reverse transcription PCR (RT-PCR), and Southern blot analysis. The fatty acid composition of soybean seeds was analyzed by gas chromatography. The 18:3 content in the transgenic generation T 1 seeds was increased 7-fold in Pb-con:PfFAD3-1, 4-fold in Pphas : PfFAD3-1, and 1.6-fold in P35S:PfFAD3-1 compared to the 18:3 content in soybean "Kwangankong". The increased content of 18:3 in the Pb-con:PfFAD3-1 soybean (T 1 ) resulted in a 52.6% increase in total fatty acids, with a larger decrease in 18:1 content than 18:2 content. The increase in 18:3 content was also maintained and reached 42% in the Pphas : PfFAD3-1 transgenic generation T 2 . Investigations of the agronomic traits of 12 Pb-con:PfFAD3-1 transgenic lines (T 1 ) revealed that plant height, number of branches, nodes, pods, total seeds, and total seed weight were significantly higher in several transgenic lines than those in non-transgenic soybean. Especially, an increase in seed size was observed upon expression of the PfFAD3-1 gene with the b-conglycinin promoter, and 6%-14% higher seed lengths were measured from the transgenic lines.

Comparison of the seed nutritional composition between conventional varieties and transgenic soybean overexpressing Physaria FAD3‐1

“…The linolenic acid contents were significantly increased about 336%, 327%, and 327% in T10‐1, T11‐8, and T12‐1 lines, respectively compared with that in KA. In contrast to previous studies, 15 the oleic acid contents were not altered in PfFAD3‐1 lines. Linoleic and linolenic acids for PfFAD3‐1 lines were not within the ranges of the reference varieties.…”

“…PfFAD3‐1 transgenic lines has an intentional change to the fatty acid profile compared to KA, producing a lower content of oleic and linoleic acids, and a higher content of linolenic acid, as a consequence of the overexpression of PfFAD3‐1 15 . In the current study, the levels of linoleic and linolenic were significantly changed between KA and three PfFAD3‐1 transgenic lines, indicating that the introduced PfFAD3‐1 gene has been successfully expressed (Table 8).…”

“…It has been reported that three omega‐3 fatty‐acid desaturase 3 ( FAD3 ) genes are expressed in soybean seeds to control ALA content 13,14 . Recently, transgenic soybean lines overexpressing the PfFAD3‐1 gene from Physaria fendleri using the β‐conglycinin promoter were successfully developed 15 . The content of ALA was increased seven‐fold in the T1 seeds of PfFAD3‐1 soybeans compared to that of the parental plant, resulting in a 52.6% increase in total fatty acids.…”

Biofortification of Cereals and Pulses Using New Breeding Techniques: Current and Future Perspectives

“…However, in most cases, it is difficult to find the desired trait in distant relatives. Hence, it is almost impossible to improve that trait through conventional breeding, i.e., improving Se contents in wheat and improving oleic and linoleic acid in the soybean (156,157). The unavailability of TOI in a species and its wild relatives is overcome through transgenic breeding.…”

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa, and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology

“…Those reports also suggested that different plant embryos have various concentrations of tolerance to unusual fatty acids. Recently, seed-specific overexpression of Lesquerella FAD3 dramatically increased the C18:3 content and increased the seed size in soybean [127], while heterologous seed-specific overexpression of CsaFAD7 in the A. thaliana embryo showed the reverse phenotype. Those reports and findings imply that the production and accumulation of C18:3 showed various tolerance in A. thaliana and soybean.…”