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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 β-conglycinin (β) or CaMV-35S (35S) promoter to generate  β-PAC  and  35S-PAC  constructs, respectively. A total of 37 transgenic lines (19 for  β-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  β-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  β-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  β-PAC  transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-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 β-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.

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