Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the beta-conglycinin (beta) or CaMV-35S (35S) promoter to generate beta-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for beta-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for beta-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the beta-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T-2 generation plants revealed that the best line among the beta-PAC transgenic seeds accumulated 146 mu g/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 mu g/g (77%) was beta-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high beta-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.